EP4384635A1 - In-situ-epitranscriptomische profilierung - Google Patents

In-situ-epitranscriptomische profilierung

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Publication number
EP4384635A1
EP4384635A1 EP22762198.4A EP22762198A EP4384635A1 EP 4384635 A1 EP4384635 A1 EP 4384635A1 EP 22762198 A EP22762198 A EP 22762198A EP 4384635 A1 EP4384635 A1 EP 4384635A1
Authority
EP
European Patent Office
Prior art keywords
probe
rna
complementary
oligonucleotide
probes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22762198.4A
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English (en)
French (fr)
Inventor
Xiao Wang
Hu ZENG
Jingyi Ren
Jiakun TIAN
Jianting GUO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Massachusetts Institute of Technology
Broad Institute Inc
Original Assignee
Massachusetts Institute of Technology
Broad Institute Inc
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Filing date
Publication date
Application filed by Massachusetts Institute of Technology, Broad Institute Inc filed Critical Massachusetts Institute of Technology
Publication of EP4384635A1 publication Critical patent/EP4384635A1/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • RNA-modifying enzymes are vital to higher eukaryotes.
  • RNA-modifying pathways have been identified to regulate cell fate, organ development, and human disease (e.g., cancer) (Jonkhout, N. et al., The RNA modification landscape in human disease. RNA 23, 1754 (2017); Li, X. et al., Epitranscriptome sequencing technologies: decoding RNA modifications. Nature Methods 14, 23 (2017)).
  • RNA modifications impact cellular functions in complex biological systems, however, is still limited. This is because traditional analyses of RNA modifications rely on mixtures of millions of cells using bulk RNA sequencing or mass spectroscopy (Li, X. et al., Epitranscriptome sequencing technologies: decoding RNA modifications. Nature Methods 14, 23 (2017)). Yet, recent studies by single-cell sequencing approaches have revealed that multicellular organisms consist of diverse cell types, and even apparently homogenous cell populations have variable single-cell states. Moreover, the same type of RNA modification may have opposing regulatory effects dependent on cell type and physiological context.
  • RNA modifications e.g., N 6 -methyladenosine (m 6 A)
  • RNA-binding proteins e.g., enzymes that install epitranscriptomic RNA modifications
  • the methods, probes, compositions, and systems disclosed herein are broadly applicable to the profiling of various epitranscriptomic RNA modifications or interactions between RNAs and RNA-binding proteins in various tissues. These methods and systems may also be useful for studying how epitranscriptomic RNA modifications or RNA-binding proteins in various cell types interact to define cell states and, for example, regulate brain function (Widagdo, J. et al., The m 6 A- epitranscriptomic signature in neurobiology: from neurodevelopment to brain plasticity. Journal of Neurochemistry 147, 137 (2016)).
  • the methods, compositions, and systems described herein may also be useful for establishing new principles of post-transcriptional gene regulation mechanisms at single-cell or subcellular resolution (approximately 150-400 nm spatial resolution, depending on both the size of DNA amplicons and optical limits) in complex biological systems, as well as for the discovery of the roles of RNA chemical fingerprints (epitranscriptomic RNA modifications) in health and disease.
  • the methods, compositions, and systems described herein may also be used on cells present within an intact tissue (e.g., a tissue sample provided by or from a human or non-human subject, such as a biopsy).
  • the present disclosure provides methods, compositions, and systems for profiling epitranscriptomic RNA modifications in a cell or multiple cells (see, for example, FIGs. 1, 10A, and 12A).
  • a cell may be contacted with one or more pairs of probes or sets of probes, which are described further herein and may be used to amplify (e.g., by rolling circle amplification) epitranscriptomically modified RNAs of interest to produce one or more concatenated amplicons.
  • RNAs of interest comprising at least one epitranscriptomic modification may be profiled, and spatiotemporal information may be obtained to improve the understanding of how epitranscriptomic modification, subcellular location, and timing affect cellular function in health and disease.
  • the methods and systems may be useful for comparing epitranscriptomic RNA modification in, for example, a cell (or multiple cells) from diseased and healthy tissue samples; or for comparing epitranscriptomic RNA modification in, for example, a cell treated with an agent (e.g., a therapeutic agent or potential therapeutic agent, such as a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate) and an untreated cell, or a diseased cell and a healthy cell.
  • an agent e.g., a therapeutic agent or potential therapeutic agent, such as a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate
  • the present disclosure provides methods for profiling epitranscriptomic RNA modifications in a cell comprising the steps of: a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe (z.e., the “padlock probe”), a second probe (z.e., the “splint probe”), and a third probe (z.e., the “primer probe”) wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein), an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide
  • the present disclosure provides methods for profiling epitranscriptomic RNA modification in a cell comprising the steps of: a) contacting the cell with one or more pairs of probes, wherein each pair of probes comprises a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) ligating the 5' end and the 3' end of the first probe together to produce
  • RNAs of interest modified with one or more epitranscriptomic modifications of interest within a cell or population of cells (e.g., in an intact tissue), or within organelles of a cell.
  • more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 RNAs of interest are profiled simultaneously using the methods described herein.
  • the epitranscriptomic RNA modification is N 6 -methyladenosine (m 6 A), N 1 -methyladenosine (m x A), pseudouridine, N 6 ,2'-O-dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 - acetylcytidine (ac 4 C), 2'-O-methylation (Nm), or 5-methylcytosine (m 5 C).
  • the present disclosure provides methods, probes, compositions, and systems for profiling interactions between RNA-binding proteins and RNAs in a cell or multiple cells, or in subcellular locations such as one or more particular organelles (see, for example, FIGs. 13A and 13C).
  • a cell may be contacted with one or more pairs of probes or sets of probes, which are described further herein and may be used to amplify (e.g., by rolling circle amplification) RNAs of interest that are bound by RNA-binding proteins to produce one or more concatenated amplicons.
  • the one or more concatenated amplicons may then be embedded in a polymeric matrix and sequenced to determine the identity of the transcripts and their location within the polymeric matrix (e.g., through SEDAL sequencing (Sequencing with Error-reduction by Dynamic Annealing and Ligation) as described further herein).
  • SEDAL sequencing Sequenced with Error-reduction by Dynamic Annealing and Ligation
  • RNAs of interest bound by at least one RNA-binding protein may be profiled, and spatiotemporal information may be obtained to improve the understanding of how interactions between RNA-binding proteins and RNAs, the subcellular location of such interactions, and the timing of such interactions affect cellular function in health and disease.
  • the methods and systems may be useful for comparing interactions between RNA-binding proteins and RNAs in, for example, a cell (or multiple cells) from diseased and healthy tissue samples; or for comparing interactions between RNA-binding proteins and RNAs in, for example, a cell treated with an agent (e.g., a therapeutic agent or potential therapeutic agent) and an untreated cell, or a diseased cell and a healthy cell.
  • an agent e.g., a therapeutic agent or potential therapeutic agent
  • the present disclosure provides methods for profiling interactions between an RNA-binding protein and one or more RNAs of interest in a cell, the method comprising: a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe (z.e., the “padlock probe”), a second probe (z.e., the “splint probe”), and a third probe (z.e., the “primer probe”) wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein), an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an oligonucleotide
  • RNA-binding proteins e.g., enzymes that install epitranscriptomic RNA modifications
  • the methods, compositions, and systems described herein may be useful for studying epitranscriptomic RNA modification and interactions between RNA-binding proteins and RNAs in tissues (e.g., developing tissues, normal tissues, diseased tissues, treated tissues), for diagnosing and treating various diseases, for research purposes, and for drug discovery.
  • the present disclosure provides methods for diagnosing a disease or disorder in a subject.
  • the methods for profiling epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs described herein may be performed on a cell, or on multiple cells, taken from a subject (e.g., a subject who is thought to have or is at risk of having a disease or disorder, or a subject who is healthy or thought to be healthy).
  • RNA-binding protein The expression of various epitranscriptomically modified RNAs of interest or RNAs of interest bound by an RNA-binding protein in the cell can then be compared to the expression of the same modified RNAs of interest in a non-diseased cell or a cell from a non-diseased tissue sample (e.g., a cell from a healthy individual, or multiple cells from a population of healthy individuals). Any difference in the epitranscriptomic RNA modification profile of the cell or the profile of interactions between RNA-binding proteins and RNAs in the cell (including of a single RNA or of multiple RNAs of interest, e.g., a specific disease signature) relative to one or more non-diseased cells may indicate that the subject has the disease or disorder.
  • Epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in one or more non-diseased cells may be profiled alongside expression in a diseased cell as a control experiment.
  • Epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in one or more non-diseased cells may have also been profiled previously, and the profile of a diseased cell may be compared to this reference data for a non-diseased cell.
  • the present disclosure provides methods of screening for an agent capable of modulating epitranscriptomic modification of one or more RNAs of interest or interactions between RNA-binding proteins and one or more RNAs of interest.
  • the methods for profiling epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs described herein may be performed in a cell in the presence of one or more candidate agents.
  • RNA-binding proteins and RNAs in the cell can then be compared to the expression of the same modified RNAs of interest or RNAs bound by an RNA-binding protein in a cell that was not exposed to the one or more candidate agents. Any difference in the epitranscriptomic RNA modification profile or the profile of interactions between RNA- binding proteins and RNAs relative to the cell that was not exposed to the candidate agent(s) may indicate that epitranscriptomic modification of the one or more RNAs of interest is modulated by the candidate agent(s).
  • a particular signature (e.g., of multiple epitranscriptomically modified RNAs of interest, or interactions between an RNA- binding protein and multiple RNAs of interest) that is known to be associated with the treatment of a disease may be used to identify agents capable of modulating epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in a desired manner and thus treating a disease.
  • the methods and systems described herein may also be used to identify drugs that have certain side effects, for example, by looking for specific epitranscriptomic RNA modification signatures or RNA-binding protein-RNA interaction signatures when one or more cells is treated with a candidate agent or known drug (or combinations of multiple candidate agents and/or known drugs, e.g., as provided in a screening library of compounds).
  • the methods and systems described herein may also be used to identify research reagents or chemical probes that may be useful for studying the basic biology of epitranscriptomic modification or interactions between RNA-binding proteins and RNAs.
  • the present disclosure provides methods for treating a disease or disorder in a subject.
  • the methods and systems for profiling epitranscriptomic RNA modification or interactions between RNA-binding proteins and RNAs described herein may be performed in a cell from a sample taken from a subject (e.g.. a subject who is thought to have or is at risk of having a disease or disorder).
  • the epitranscriptomic RNA modification profile or profile of interactions with RNA-binding proteins of one or more RNAs of interest in the cell can then be compared to the epitranscriptomic RNA modification profile or profile of interactions with RNA-binding proteins of one or more RNAs of interest in a cell from a non-diseased tissue sample.
  • a treatment for the disease or disorder may then be administered to the subject if any difference in the epitranscriptomic RNA modification profile or the profile of interactions between RNA-binding proteins and RNAs relative to a non-diseased cell is observed.
  • Epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in one or more non-diseased cells may be profiled alongside epitranscriptomic RNA modification in a diseased cell as a control experiment.
  • RNA-binding proteins and RNAs in one or more non-diseased cells may have also been profiled previously, and epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in a diseased cell may be compared to this reference data for a non-diseased cell.
  • the present disclosure provides pairs of probes comprising a first probe (also referred to herein as the “padlock” probe) and a second probe (also referred to herein as the “primer” probe), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe.
  • the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a
  • the present disclosure provides sets of probes comprising a first probe (z.e., the “padlock probe”), a second probe (z.e., the “splint probe”), and a third probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe
  • the present disclosure provides sets of probes comprising a first probe (z.e., the “padlock probe”), a second probe (z.e., the “splint probe”), and a third probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe.
  • kits e.g., a kit comprising any of the pairs of probes or sets of probes disclosed herein.
  • the kit comprises multiple pairs of probes or sets of probes as described herein, each of which can be used to identify a specific epitranscriptomically modified RNA of interest or interaction between a particular RNA-binding protein and RNA of interest.
  • the kit comprises more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 pairs of probes.
  • the kit comprises more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 sets of probes.
  • the kits described herein may also include any other reagents or components useful in performing the methods described herein, including, but not limited to, cells, enzymes such as a ligase and/or a polymerase, amine-modified nucleotides, agents that bind an RNA modification (e.g..).
  • primary antibodies secondary antibodies, proteins, peptides, aptamers, small molecules, etc.
  • buffers reagents (including dyes, stains, buffers, and more), and monomers for making a polymeric matrix (e.g.. a polyacrylamide matrix).
  • reagents including dyes, stains, buffers, and more
  • monomers for making a polymeric matrix e.g. a polyacrylamide matrix
  • the present disclosure provides systems for profiling epitranscriptomic RNA modifications in a cell.
  • a system comprises: a) a cell (e.g., an isolated cell, or a cell present within an intact tissue); b) one or more pairs of probes comprising a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; c)
  • the present disclosure provides systems comprising: a) a cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope; and d) a computer.
  • the first probe comprises an oligon
  • the present disclosure provides systems comprising: a) a cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope; and d) a computer.
  • the first probe comprises an oligonucle
  • any of the probes (z.e., the pairs of probes and sets of probes) described herein may be used in the systems contemplated by the present disclosure.
  • FIG. 1 provides an outline of a method for profiling of m 6 A-modified RNAs in situ.
  • a secondary anti-m 6 A antibody is conjugated to a polymerizable DNA primer, which anneals to a padlock probe that resides on the same RNA.
  • Functionalized cDNA amplicons are then generated from the co-localized DNA primer and padlock probes.
  • Chemically functionalized cDNA amplicons are then covalently linked to a polyacrylamide matrix to allow tissue optical clearing and biomolecule processing.
  • FIGs. 2A-2D show an example of single-cell in situ profiling of m 6 A RNA modification.
  • FIG. 2A shows detection of m 6 A-modified beta-actin RNAs.
  • FIGs. 2B-2C provide several negative controls, no primary antibody (FIG. 2B), secondary antibody incubation with no DNA conjugated (FIG. 2C), and no secondary antibody (FIG. 2D).
  • FIGs. 3A-3D are images showing methods for resolving RNA modification mediated gene regulation in biological tissues by enabling single-cell in situ epitranscriptomic profiling.
  • FIG. 3A shows various chemical modifications on eukaryotic messenger RNA. The collection of all the modification sites is termed the epitranscriptome.
  • FIG. 3A shows various chemical modifications on eukaryotic messenger RNA. The collection of all the modification sites is termed the epitranscriptome.
  • FIG. 3B provides a schematic showing single-cell heterogeneity of epitranscriptomic states across different cell types, states, and subcellular locations. These questions have not been addressed by previously developed bulk transcriptomic or epitranscriptomic analysis methods.
  • FIG. 3C shows three-dimensional (3D) in situ sequencing of RNAs and RNA modifications. DNA- conjugated modification- specific binders are used together with RNA-sequence-specific DNA probes that hybridize to cellular mRNAs to selectively visualize modified RNAs in intact tissue. Each RNA-sequence-specific probe contains a barcode encoding identity of the gene, which is read-out through image-based in situ sequencing.
  • FIG. 3D shows resolving RNA modification-mediated gene regulation in tissue as exemplified by the four possible changes of RNA modifications during brain activity.
  • the shift of epitranscriptomic patterns, along with gene expression changes of RNA-modifying pathways, provides information on possible gene regulation schemes in different cells and brain regions.
  • In situ epitranscriptomic sequencing can also be combined with precise genetic perturbation to fully dissect gene regulation mechanisms.
  • FIG. 4 provides a schematic showing questions regarding RNA modifications that cannot be anwsered by bulk epitranscriptomic sequencing methods.
  • FIG. 5 shows examples of mutagenic and non-mutagenic epitranscriptomic RNA modifications.
  • FIG. 6 provides a schematic showing the role of m 6 A in brain function.
  • m 6 A modification is reversible and recognized by m 6 A-binding proteins, which regulate multiple steps of the mRNA life cycle.
  • m 6 A-protein complexes have been found to be enriched in synapses. Alteration of the m 6 A pathway impacts multiple physiological functions and is associated with a spectrum of psychiatric disorders.
  • FIG. 7 provides a schematic of the 3D-m 6 A-seq method.
  • DNA-conjugated m 6 A-specific binders together with RNA-sequence-specific DNA probes will hybridize to cellular mRNAs within the intact tissue. Only m 6 A-modified sites are enzymatically replicated as cDNA amplicons, while non-modified RNAs are not amplified.
  • Each RNA-sequence-specific probe contains a barcode encoding the identity of the gene and the m 6 A site, which is read-out through in situ sequencing (SEDAL).
  • FIG. 8 provides schematics of four exemplary biological investigations that may be carried out using single-cell in situ sequencing of RNA modifications.
  • FIGs. 9A-9D show the background and applications of spatial epitranscriptomics.
  • FIG. 9A shows that m 6 A and its associated RNA-binding proteins (RBPs) regulate various important pathways.
  • FIGs. 9B and 9C show knowledge gaps in epitranscriptomics that the methods provided herein are applicable to elucidating.
  • FIG. 9D provides a schematic of in situ single-cell epitranscriptomics.
  • FIGs. 10A-10E show the design and principles of m 6 A-map vl.
  • FIG. 10A shows the workflow of m 6 A-map vl.
  • FIG. 10B shows the conjugation strategy used to synthesize the PAPG-oligo.
  • FIG. 10C demonstrates the detection of actin P (ACTB) site 1217 m 6 A in HeEa cells via antibody-PAPG-oligo detection, showing a 20-30 fold signal enrichment over negative control.
  • FIG. 10D demonstrates the detection of metastasis associated lung adenocarcinoma transcript 1 (MAEAT1) site 2601 m 6 A in HeEa cells via antibody- independent biotinylated-YTH-streptavidin-oligo detection, showing no enrichment over negative control.
  • FIG. 10E demonstrates the detection of MALAT1 site 1248 m 6 A in mouse hippocampus via antibody-PAPG-oligo detection, showing an approximately 15-fold signal enrichment over negative control.
  • FIGs. 11A-11B show substitution of a secondary antibody for PAPG.
  • FIG. 11A shows the labeling chemistry used by SiteClick antibody labeling kit (subsequent conjugation with alkyne-oligo can be performed using click chemistry).
  • FIG. 11B demonstrates the detection of ACTB site 1217 m 6 A in HeEa cells via antibody-PAPG-oligo detection and antibody-antibody-oligo detection. The antibody-antibody-oligo detection scheme showed much lower signal enrichment over IgG control groups.
  • FIGs. 12A-12D show the design and principles of m 6 A-map v2.
  • FIG. 12A shows the workflow of m 6 A-map v2, achieving simultaneous detection of m 6 A-methylated RNAs and their non-methylated counterparts.
  • FIG. 12B demonstrates the “winner-takes-all” behavior of the probes targeting the same RNA: when two pairs of STARmap probes (see PCT publication number WO 2019/199579, which is incorporated herein by reference) target the same ACTB mRNA in HeEa cells, only one of them will get amplified at each ACTB mRNA molecule.
  • FIG. 12A shows the workflow of m 6 A-map v2, achieving simultaneous detection of m 6 A-methylated RNAs and their non-methylated counterparts.
  • FIG. 12B demonstrates the “winner-takes-all” behavior of the probes targeting the same RNA: when two pairs of STARmap probes (see PCT publication number WO 2019/199579,
  • FIG. 12C demonstrates the detection of MALAT1 site 2601 m 6 A in HeLa cells via m 6 A-map v2, showing higher m 6 A stoichiometry in M and early G1 phases.
  • FIG. 12D demonstrates the detection of ACTB site 1217 m 6 A in HeLa cells via m 6 A-map v2, showing higher m 6 A stoichiometry in non-dividing cells.
  • FIGs. 13A-13C show RBP-map principles and experimental results.
  • FIG. 13A shows the workflow for single RBP-RNA interaction mapping.
  • FIG. 13B shows that HeLa cells transfected with 3xFLAG-YTH(WT/mut)-T2A-mCherry were tested for YTH binding of ACTB via the workflow shown in FIG. 13 A.
  • FIG. 13C shows the workflow for multiplexed RBP-RNA mapping.
  • Each antibody is conjugated to a unique DNA primer with a corresponding gap-filling probe annealed to it.
  • the three-part probes are hybridized to mRNAs.
  • a mixture of different antibodies targeting different RBPs are then added to the sample, followed by ligation and RCA. Barcode information on the probe can be read out through in situ sequencing.
  • FIG. 14 provides representative images of the first round of sequencing in an m 6 A- map 100-gene data collection experiment. ChOl and ch04 correspond to STARmap amplicons, while ch02 and ch03 correspond to m 6 A amplicons.
  • FIGs. 15A-15H show quality control and overview of statistics of m 6 A-map vl 100- gene dataset.
  • FIG. 15A shows the average number of reads per cell for each gene.
  • FIG. 15A shows the average number of reads per cell for each gene.
  • FIG. 15B shows the signal-to-noise ratio (calculated using the ratio of anti-m 6 A signal vs. IgG) in each well.
  • FIG. 15C shows the detected genes and reads per cell in each well.
  • FIG. 15D shows the correlation of estimated relative m 6 A stoichiometry between well A2 and well B3.
  • FIG. 15E shows the correlation between estimated m 6 A stoichiometry and STARmap reads.
  • FIG. 15F shows the relative m 6 A stoichiometry for each gene measured in each well.
  • FIG. 15G provides a comparison of estimated m 6 A stoichiometry by m 6 A-map vl vs. reported m 6 A stoichiometry of representative loci.
  • FIG. 15H provides a scatter plot of the data shown in FIG. 15G.
  • FIGs. 16A-16C show subcellular analysis and cell cycle analysis of m 6 A-map vl 100-gene dataset.
  • FIG. 16A shows the effect of m 6 A deposition on RNA subcellular localization (x-axis: the log2 fold-change of nuclear percentage of m 6 A deposited portion vs. the non-m 6 A deposited portion of a given RNA; y-axis: -logio of p value).
  • FIG. 16B shows that the cell cycle was determined using FUCCI fluorescent intensity.
  • FIG. 16C shows representative genes with cell-cycle dependent m 6 A fluctuations.
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a treatment or therapeutic agent, or a composition of treatments or therapeutic agents, in or on a subject.
  • amplicon refers to a nucleic acid (e.g., RNA) that is the product of an amplification reaction (z.e., the production of one or more copies of a genetic fragment or target sequence) or replication reaction. Amplicons can be formed artificially using, for example, PCR or other polymerization reactions.
  • amplification reaction z.e., the production of one or more copies of a genetic fragment or target sequence
  • replication reaction z.e., the production of one or more copies of a genetic fragment or target sequence
  • Concatenated amplicons refers to multiple amplicons that are joined together to form a single nucleic acid molecule.
  • Concatenated amplicons can be formed, for example, by rolling circle amplification (RCA), in which a circular oligonucleotide is amplified to produce multiple linear copies of the oligonucleotide as a single nucleic acid molecule comprising multiple amplicons that are concatenated.
  • RCA rolling circle amplification
  • an “antibody” refers to a glycoprotein belonging to the immunoglobulin superfamily.
  • the terms antibody and immunoglobulin are used interchangeably.
  • mammalian antibodies are typically made of basic structural units each with two large heavy chains and two small light chains.
  • Five different antibody isotypes are known in mammals (IgG, IgA, IgE, IgD, and IgM), which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.
  • antibody as used herein also encompasses antibody fragments, nanobodies, and single chain antibodies, as well as variants of antibodies.
  • antibody variants may also be used to encompass antibody fragments.
  • an antibody or antibody variant is administered as a treatment for a disease or disorder (e.g., one that is associated with a change in the profile of epitranscriptomic RNA modifications in a cell taken from a subject).
  • an antibody is conjugated to an oligonucleotide probe as described herein.
  • the antibody binds to an epitranscriptomic RNA modification (e.g., the antibody is an anti-m 6 A antibody, an anti-m 1 A antibody, an anti- pseudouridine antibody, an anti-m 6 Am antibody, an anti-m 7 G antibody, an anti-ac 4 C antibody, an anti-Nm antibody, or an anti-m 5 C antibody).
  • an epitranscriptomic RNA modification e.g., the antibody is an anti-m 6 A antibody, an anti-m 1 A antibody, an anti- pseudouridine antibody, an anti-m 6 Am antibody, an anti-m 7 G antibody, an anti-ac 4 C antibody, an anti-Nm antibody, or an anti-m 5 C antibody.
  • a “cell,” as used herein, may be present in a population of cells (e.g., in a tissue, a sample, a biopsy, an organ, or an organoid).
  • a population of cells is composed of a plurality of different cell types.
  • Cells for use in the methods and systems of the present disclosure can be present within an organism, a single cell type derived from an organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, cells from a subject, etc. Virtually any cell type and size can be accommodated in the methods and systems described herein.
  • the cells are mammalian cells (e.g., complex cell populations such as naturally occurring tissues).
  • the cells are from a human.
  • the cells are collected from a subject (e.g., a human) through a medical procedure, such as a biopsy.
  • the cells may be a cultured population (e.g., a culture derived from a complex population or a culture derived from a single cell type where the cells have differentiated into multiple lineages).
  • the cells may also be provided in situ in a tissue sample.
  • Cell types contemplated for use in the methods and systems of the present disclosure include, but are not limited to, stem and progenitor cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc.), endothelial cells, muscle cells, myocardial cells, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells, hematopoietic cells, lymphocytes such as T-cells (e.g., Thl T cells, Th2 T cells, ThO T cells, cytotoxic T cells) and B cells (e.g., pre-B cells), monocytes, dendritic cells, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, immune cells, neurons, hepatocytes, and cells involved with particular organs (e.g., thymus, endocrine glands, pancreas, brain, neurons, glia, astrocytes, dend
  • the cells may also be transformed or neoplastic cells of different types (e.g., carcinomas of different cell origins, lymphomas of different cell types, etc.) or cancerous cells of any kind (e.g., from any of the cancers disclosed herein).
  • Cells of different origins e.g., ectodermal, mesodermal, and endodermal
  • the cells are microglia, astrocytes, oligodendrocytes, excitatory neurons, or inhibitory neurons.
  • the cells are HeLa cells.
  • cells of multiple cell types are present within the same sample.
  • complementar is used herein to refer to two oligonucleotide sequences (e.g., DNA or RNA) comprising bases that hydrogen bond to one another.
  • the degree of complementarity between two oligonucleotide sequences can vary, from complete complementarity to no complementarity (e.g., 100% complementarity, 99% complementarity, 98% complementarity, 97% complementarity, 96% complementarity, 95% complementarity, 90% complementarity, 85% complementarity, 80% complementarity, or less than 80% complementarity).
  • two oligonucleotide sequences may be only partially complementary to one another (e.g., in the probes described herein, wherein only a portion of the probe is complementary to another probe, or to an RNA of interest).
  • a sequence is complementary to only a portion of another sequence.
  • a sequence is complementary to another sequence under certain conditions (e.g., certain salt concentrations, pHs, etc.).
  • Epitranscriptomic modification include any biochemical modification of an RNA within a cell. Such modifications can be chemical modifications, including, for example, methylation of a nucleotide at various positions.
  • Chemical epitranscriptomic modifications include, but are not limited to, N 6 -methyladenosine (m 6 A), N 1 - methyladenosine (m x A), pseudouridine, N 6 ,2'-O-dimethyladenosine (m 6 Am), 7- methylguanosine (m 7 G), N 4 -acetylcytidine (ac 4 C), 2'-O-methylation (Nm), and 5- methylcytosine (m 5 C).
  • an epitranscriptomic modification is N 6 - methyladenosine (m 6 A).
  • RNA modifications include adenosine-to-inosine mutations and queuosine (z.e., queuosine replaces another nucleotide in the RNA).
  • Various types of cellular RNA may be epitranscriptomically modified, including, but not limited to, ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • Other epitranscriptomic modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, E. M. et al. Nature. 541, 339-346 (2017).
  • polynucleotide refers to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA and mean any chain of two or more nucleotides.
  • the polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, and single-stranded or double- stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc.
  • An “aptamer” is a type of oligonucleotide molecule that binds to a specific target molecule (e.g., an epitranscriptomic modification).
  • a “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds.
  • the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long.
  • a protein may refer to an individual protein or a collection of proteins. Proteins may contain only natural amino acids, although non-natural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification.
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, synthetic, or any combination of these.
  • a protein may also be a therapeutic protein administered as a treatment for a disease or disorder (e.g., one that is associated with a change in the profile of epitranscriptomic RNA modification in a cell taken from a subject).
  • the protein is an antibody, or an antibody variant (including antibody fragments).
  • a protein binds to an epitranscriptomic RNA modification (e.g., an m 6 A-specific YTH domain protein).
  • RNA-binding protein refers to any protein that is capable of binding RNA, or an epitranscriptomic modification of an RNA.
  • RNA-binding proteins may be proteins that introduce an epitranscriptomic modification onto an RNA.
  • RNA-binding proteins may also be proteins that recognize and bind to an epitranscriptomic modification of an RNA.
  • the RNA-binding protein comprises a YTH family protein (e.g., YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2), an IGF2BP family protein (e.g., IGF2BP1, IGF2BP2, or IGF2BP3), or FMRI.
  • RNA-binding proteins that can be profiled using the methods provided herein also include, but are not limited to, those disclosed in Wang, X. et al., N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell 161, 1388-1399, doi:10.1016/j.cell.2015.05.014 (2015); Wang, X. et al., N6- methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117-120, doi:10.1038/naturel2730 (2014); Shi, H. et al., YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA.
  • YTHDC1 Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell 61, 507-519, doi:10.1016/j.molcel.2016.01.012 (2016); Roundtree, I. A. et al., YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife 6, doi:10.7554/eLife.31311 (2017); Hsu, P. J. et al., Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis.
  • RNA transcript is the product resulting from RNA polymerase- catalyzed transcription of a DNA sequence.
  • primary transcript When the RNA transcript is a complimentary copy of the DNA sequence, it is referred to as the primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA)” refers to the RNA that is without introns and can be translated into polypeptides by the cell.
  • sample refers to any sample including tissue samples (such as tissue sections, surgical biopsies, and needle biopsies of a tissue); cell samples; or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise).
  • tissue samples such as tissue sections, surgical biopsies, and needle biopsies of a tissue
  • cell samples such as cell fractions, fragments, or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise).
  • biological samples include, but are not limited to, blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample.
  • a biological sample is a surgical biopsy taken from a subject, for example, a biopsy of any of the tissues described herein.
  • a biological sample is a tumor biopsy.
  • the sample is brain tissue.
  • the tissue is cardiac tissue.
  • the sample is epithelial tissue, connective tissue, muscular tissue, or nervous tissue.
  • the sample is tissue from the central nervous system (e.g., brain).
  • the cells used in the methods described herein come from such a sample or biological sample.
  • a “subject” to which administration is contemplated refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey) or mouse).
  • the term “patient” refers to a subject in need of treatment of a disease.
  • the subject is human.
  • the patient is human.
  • the human may be a male or female at any stage of development.
  • a subject or patient “in need” of treatment of a disease or disorder includes, without limitation, those who exhibit any risk factors or symptoms of a disease or disorder.
  • a subject is a non-human experimental animal (e.g., a mouse, rat, dog, or non-human primate).
  • the term “therapeutic agent,” as used herein, refers to any agent that can be used to treat a disease or disorder, or reduce or alleviate the symptoms of a disease or disorder.
  • the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate.
  • the therapeutic agent is a known drug and/or an FDA-approved drug.
  • the protein is an antibody.
  • the protein is an antibody variant.
  • the protein is a receptor, or a fragment or variant thereof.
  • the protein is a cytokine.
  • the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).
  • a “therapeutically effective amount” of a treatment or therapeutic agent is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a treatment or therapeutic agent means an amount of the therapy, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • a tissue is a group of cells and their extracellular matrix from the same origin. Together, the cells carry out a specific function. The association of multiple tissue types together forms an organ. The cells may be of different cell types.
  • a tissue is an epithelial tissue. Epithelial tissues are formed by cells that cover an organ surface (e.g., the surface of the skin, airways, soft organs, reproductive tract, and inner lining of the digestive tract). Epithelial tissues perform protective functions and are also involved in secretion, excretion, and absorption.
  • a tissue is a connective tissue.
  • Connective tissues are fibrous tissues made up of cells separated by non-living material (e.g., an extracellular matrix). Connective tissues provide shape to organs and hold organs in place. Connective tissues include fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Examples of connective tissues include, but are not limited to, blood, bone, tendon, ligament, adipose, and areolar tissues.
  • a tissue is a muscular tissue.
  • Muscular tissue is an active contractile tissue formed from muscle cells. Muscle tissue functions to produce force and cause motion. Muscle tissue includes smooth muscle (e.g., as found in the inner linings of organs), skeletal muscle (e.g., as typically attached to bones), and cardiac muscle (e.g., as found in the heart, where it contracts to pump blood throughout an organism).
  • a tissue is a nervous tissue. Nervous tissue includes cells comprising the central nervous system and peripheral nervous system. Nervous tissue forms the brain, spinal cord, cranial nerves, and spinal nerves (e.g., motor neurons).
  • a tissue is brain tissue.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed (e.g., prophylactically or upon suspicion or risk of disease).
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms in the subject, or family members of the subject). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • treatment may be administered after using the methods disclosed herein and observing a change in the profile of epitranscriptomic RNA modifications in a cell or tissue in comparison to a healthy cell or tissue.
  • the present disclosure provides methods, compositions, and systems for profiling epitranscriptomic RNA modifications in a cell, or in multiple cells (e.g., cells present within an intact tissue, or isolated cells) at single-cell resolution and at subcellular resolution. Also provided by the present disclosure are methods for profiling interactions between one or more RNAs of interest in a cell and an RNA-binding protein (e.g., a protein that introduces an epitranscriptomic modification of an RNA of interest). The present disclosure also provides methods for diagnosing a disease or disorder in a subject based on a profile of epitranscriptomic RNA modifications or a profile of interactions between an RNA binding protein and RNA(s) in a cell, including cells within an intact tissue.
  • an RNA-binding protein e.g., a protein that introduces an epitranscriptomic modification of an RNA of interest.
  • the present disclosure also provides methods for treating a disease or disorder in a subject in need thereof.
  • Methods of screening for or testing a candidate agent capable of modulating epitranscriptomic modification of one or more RNAs or interactions between one or more RNA(s) and an RNA-binding protein are also provided by the present disclosure.
  • Pairs of probes and sets of probes, which may be useful for performing the methods described herein, are also provided by the present disclosure, as well as kits comprising any of the probes described herein.
  • the present disclosure provides methods for profiling epitranscriptomic RNA modifications in a cell (or in multiple cells, e.g., in an intact tissue).
  • a cell may be contacted with one or more pairs of probes or sets of probes, which are described further herein and may be used to identify and locate RNAs of interest comprising at least one epitranscriptomic modification to produce one or more concatenated amplicons.
  • the one or more concatenated amplicons may then be embedded in a polymeric matrix and sequenced to determine the identity of the transcripts and their location within the polymeric matrix (e.g., through SEDAL sequencing as described further herein).
  • epitranscriptomically-modified RNAs in a cell may be profiled.
  • the present disclosure provides methods for profiling epitranscriptomic RNA modification in a cell comprising the steps of: a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe (/'. ⁇ ?., the “padlock probe”), a second probe (i.e., the “splint probe”), and a third probe (i.e., the “primer probe”) wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein), an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification
  • the present disclosure provides methods for profiling epitranscriptomic RNA modifications in a cell (or in multiple cells, e.g., in an intact tissue) comprising the steps of: a) contacting the cell with one or more pairs of probes, wherein each pair of probes comprises a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) lig
  • the epitranscriptomic RNA modification is N 6 - methyladenosine (m 6 A), N 1 -methyladenosine (m 1 A), pseudouridine, N 6 ,2'-O- dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -acetylcytidine (ac 4 C), 2'-O- methylation (Nm), or 5-methylcytosine (m 5 C).
  • Other epitranscriptomic modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, E. M.
  • the epitranscriptomic RNA modification is an adenosine-to-inosine modification.
  • the epitran scrip tomic modification is queuosine (z.e., queuosine replaces another nucleotide in the RNA), polyadenylation, intron splicing, or histone mRNA processing.
  • the epitranscriptomic RNA modification is N 6 - methyladenosine (m 6 A).
  • a single epitranscriptomic modification may be profiled in a cell using the methods disclosed herein, or multiple different epitranscriptomic modifications (e.g., two, three, four, five, or more) may be profiled in the cell simultaneously.
  • the methods for profiling epitranscriptomic RNA modifications disclosed herein contemplate the use of a set of probes comprising a first probe, a second probe, and a third probe. Such methods utilize what is referred to herein as a “three -probe strategy.”
  • the second probe of the set of probes (also referred to herein as the “splint probe”) comprises a portion that recognizes an epitranscriptomic RNA modification (z.e., a post-transcriptional modification present on a particular RNA of interest).
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a peptide.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds the epitranscriptomic RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptomic modification through a mechanism comprising a biotin-streptavidin interaction.
  • the portion of the probe that recognizes an epitranscriptomic RNA modification may be a protein (e.g., an antibody or antibody variant, or any protein that is otherwise capable of binding to a specific epitranscriptomic modification). In certain embodiments, the protein is PAPG.
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an agent that binds an antibody, or an antibody variant.
  • the second probe may comprise a secondary antibody, and a primary antibody may be used to bind to the epitranscriptomic RNA modification. The secondary antibody on the second probe then recognizes the primary antibody bound to the epitranscriptomic RNA modification. See, for example, FIG. 1.
  • the second probe may comprise the protein PAPG, which may bind an antibody that binds to the epitranscriptomic RNA modification.
  • the method may optionally further comprise contacting the cell with an antibody that recognizes an epitranscriptomic RNA modification, wherein the antibody that recognizes an epitranscriptomic RNA modification is recognized and bound by the protein of the second probe (e.g., PAPG).
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant.
  • the method may optionally further comprise contacting the cell with a primary antibody that recognizes the epitranscriptomic RNA modification and is recognized by the secondary antibody of the second probe.
  • the cell may be contacted with the primary antibody before or after being contacted with the one or more pairs of probes. In certain embodiments, the cell is contacted with the primary antibody prior to being contacted with the one or more pairs of probes.
  • the primary antibody is an anti-m 6 A antibody, an anti-m 1 A antibody, an anti-pseudouridine antibody, an anti-m 6 Am antibody, an anti-m 7 G antibody, an anti-ac 4 C antibody, an anti-Nm antibody, or an anti-m 5 C antibody.
  • the present disclosure also contemplates the use of any agent capable of binding an epitranscriptomic RNA modification directly on the probes described herein.
  • the portion of the second probe that binds the epitranscriptomic RNA modification directly comprises a protein.
  • the protein is an m 6 A- specific YTH domain protein.
  • the portion of the second probe that binds the epitranscriptomic RNA modification directly comprises an antibody, or an antibody variant.
  • the second probe of the set of probes further comprises a polymerization blocker.
  • the polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the methods described herein.
  • the polymerization blocker is at the 3' end of the second oligonucleotide probe.
  • the polymerization blocker can be, for example, any chemical moiety that prevents a polymerase from using the second oligonucleotide probe as a primer for polymerization.
  • the polymerization blocker is a nucleic acid residue comprising a blocked 3' hydroxyl group (e.g., comprising an oxygen protecting group on the 3' hydroxyl group).
  • the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group.
  • the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added.
  • the polymerization blocker comprises an inverted nucleic acid residue.
  • the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue.
  • the polymerization blocker is an inverted thymine residue.
  • the second probe also comprises a portion that is complementary to a portion of the first probe.
  • the portions of the first and second probes that are complementary to one another may be the same on each set of probes. In some embodiments, the portions of the first and second probes that are complementary to one another are unique on each of the first and second probes.
  • the portion of the second probe that is complementary to a portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • each portion of the second probe is connected by an optional linker.
  • the optional linker is a nucleotide linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the second probe of the sets of probes used in the methods described herein comprises the structure:
  • portion recognizing epitranscriptomic RNA modification -[portion complementary to first probe]-3', wherein ]-[ comprises an optional linker (e.g., a nucleotide linker).
  • ]-[ represents a direct linkage between two portions of the second probe (z.e., a phosphodiester bond).
  • the portion recognizing an epitranscriptomic RNA modification and the oligonucleotide portion of the probe are attached to each other via click chemistry (see, for example, FIG. 10B).
  • the first probe of the sets of probes used in the methods described herein includes an oligonucleotide portion that is complementary to the second probe.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' end and the 3' end of the first probe.
  • the first probe of the sets of probes used in the methods disclosed herein also comprises an oligonucleotide portion that is complementary to an RNA of interest.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.
  • the first probe of the sets of probes used in the methods disclosed herein also comprises an oligonucleotide barcode sequence made up of a specific sequence of nucleotides.
  • the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length.
  • the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.
  • the barcodes of the oligonucleotide probes described herein may comprise genespecific sequences used to identify RNAs of interest (z.e., RNAs that have been modified with at least one particular epitranscriptomic modification).
  • the first probe comprises the structure:
  • ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • any of the oligonucleotide portions of the probes that make up the sets of probes provided herein comprise DNA.
  • the third probe of the sets of probes used in the methods provided herein comprises a portion complementary to an RNA of interest and a portion complementary to the first probe of the set of probes.
  • the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.
  • the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the portion of the third probe that is complementary to a portion of the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
  • the third probe of the set of probes comprises the structure: 5'-[portion complementary to RNA of interest] -[portion complementary to first probe] -3', wherein ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • the methods disclosed for profiling epitranscriptomic RNA modifications herein contemplate the use of a pair of probes comprising a first probe and a second probe. Such methods utilize what is referred to herein as a “two-probe strategy.”
  • the second probe of the pair of probes (also referred to herein as the “primer probe”) comprises a portion that recognizes an epitranscriptomic RNA modification (z.e., a post-transcriptional modification present on a particular RNA of interest).
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a peptide.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds the epitranscriptomic RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptomic modification through a mechanism comprising a biotin- streptavidin interaction.
  • the portion of the probe that recognizes an epitranscriptomic RNA modification may be a protein (e.g., an antibody or antibody variant, or any protein that is otherwise capable of binding to a specific epitranscriptomic modification). In some embodiments, the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an agent that binds an antibody, or an antibody variant.
  • the second probe may comprise a secondary antibody, and a primary antibody may be used to bind to the epitranscriptomic RNA modification.
  • the secondary antibody on the second probe then recognizes the primary antibody bound to the epitranscriptomic RNA modification.
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant.
  • the method may optionally further comprise contacting the cell with a primary antibody that recognizes the epitranscriptomic RNA modification and is recognized by the secondary antibody of the second probe.
  • the cell may be contacted with the primary antibody before or after being contacted with the one or more pairs of probes. In certain embodiments, the cell is contacted with the primary antibody prior to being contacted with the one or more pairs of probes.
  • the primary antibody is an anti- m 6 A antibody, an anti-m 1 A antibody, an anti-pseudouridine antibody, an anti-m 6 Am antibody, an anti-m 7 G antibody, an anti-ac 4 C antibody, an anti-Nm antibody, or an anti-m 5 C antibody.
  • the present disclosure also contemplates the use of any agent capable of binding an epitranscriptomic RNA modification on the probes described herein.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a protein.
  • the protein is PAPG.
  • the method further comprises contacting the cell with an antibody that recognizes an epitranscriptomic RNA modification, wherein the antibody that recognizes an epitranscriptomic RNA modification is recognized and bound by PAPG.
  • the protein is an m 6 A-specific YTH domain protein that can bind an epitranscriptomic RNA modification directly.
  • the portion of the second probe that binds the epitranscriptomic RNA modification binds the modification directly and comprises an antibody, or an antibody variant.
  • the second probe of the pair of probes also comprises a portion that is complementary to a portion of the first probe.
  • the portions of the first and second probes that are complementary to one another may be the same on each set of probes. In some embodiments, the portions of the first and second probes that are complementary to one another are unique on each pair of probes. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the second probe that is complementary to a portion of the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • each portion of the second probe of the pair of probes is connected by an optional linker.
  • the optional linker is a nucleotide linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the second probe used in the methods described herein comprises the structure:
  • ]-[ comprises an optional linker (e.g., a nucleotide linker).
  • ]-[ represents a direct linkage between two portions of the second probe (z.e., a phosphodiester bond).
  • the first probe of the pair of probes used in the methods described herein includes an oligonucleotide portion that is complementary to the second probe.
  • the portion of the first probe that is complementary to a portion of the second probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the first probe that is complementary to a portion of the second probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' end and the 3' end of the first probe.
  • the first probe of the pair of probes used in the methods disclosed herein also comprises an oligonucleotide portion that is complementary to an RNA of interest.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.
  • the first probe of the pair of probes used in the methods disclosed herein also comprises an oligonucleotide barcode sequence made up of a specific sequence of nucleotides.
  • the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length.
  • the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.
  • the barcodes of the oligonucleotide probes described herein may comprise genespecific sequences used to identify RNAs of interest (z.e., RNAs that have been modified with at least one particular epitranscriptomic modification).
  • the first probe comprises the structure:
  • ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • any of the oligonucleotide portions of the first probe and/or the second probe comprise DNA.
  • the present disclosure provides methods for profiling interactions between an RNA-binding protein and one or more RNAs of interest in a cell, the method comprising: a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide
  • RNA-binding protein is a protein that introduces an epitranscriptomic modification onto an RNA.
  • the RNA-binding protein comprises a YTH family protein (e.g., YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2), an IGF2BP family protein (e.g., IGF2BP1, IGF2BP2, or IGF2BP3), or FMRI.
  • YTH family protein e.g., YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2
  • IGF2BP family protein e.g., IGF2BP1, IGF2BP2, or IGF2BP3
  • FMRI FMRI
  • the methods for profiling interactions between RNA-binding proteins and RNAs of interest disclosed herein contemplate the use of a set of probes comprising a first probe, a second probe, and a third probe.
  • the second probe of the set of probes (also referred to herein as the “splint probe”) comprises a portion that recognizes an RNA-binding protein (e.g., an enzyme that introduces an epitranscriptomic modification onto an RNA).
  • the portion of the second probe that binds the RNA-binding protein comprises a peptide.
  • the portion of the second probe that binds the RNA-binding protein comprises an aptamer.
  • the portion of the second probe that binds the RNA-binding protein comprises a small molecule. In certain embodiments, the second probe binds to the RNA-binding protein through a mechanism comprising a biotin-streptavidin interaction.
  • the portion of the probe that recognizes an RNA-binding protein may be a protein (e.g., an antibody or antibody variant, or any protein that is otherwise capable of binding to a specific RNA-binding protein).
  • the portion of the second probe that recognizes the RNA-binding protein comprises an agent that binds an antibody, or an antibody variant.
  • the second probe may comprise a secondary antibody, and a primary antibody may be used to bind to the RNA-binding protein.
  • the portion of the second probe that recognizes the RNA-binding protein comprises an antibody (e.g., a secondary antibody), or an antibody variant.
  • the method may optionally further comprise contacting the cell with a primary antibody that recognizes the RNA-binding protein and is recognized by the secondary antibody of the second probe. The cell may be contacted with the primary antibody before or after being contacted with the one or more pairs of probes.
  • the cell is contacted with the primary antibody prior to being contacted with the one or more pairs of probes.
  • the present disclosure also contemplates the use of any agent capable of recognizing and directly binding an RNA-binding protein on the probes described herein.
  • the portion of the second probe that binds the RNA-binding protein directly comprises a protein.
  • the portion of the second probe that binds the RNA-binding protein directly comprises an antibody, or an antibody variant.
  • the second probe of the set of probes further comprises a polymerization blocker.
  • the polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the methods described herein.
  • the polymerization blocker is at the 3' end of the second oligonucleotide probe.
  • the polymerization blocker can be, for example, any chemical moiety that prevents a polymerase from using the second oligonucleotide probe as a primer for polymerization.
  • the polymerization blocker is a nucleic acid residue comprising a blocked 3' hydroxyl group (e.g., comprising an oxygen protecting group on the 3' hydroxyl group).
  • the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.
  • the second probe also comprises a portion that is complementary to a portion of the first probe.
  • the portions of the first and second probes that are complementary to one another may be the same on each set of probes. In some embodiments, the portions of the first and second probes that are complementary to one another are unique on each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is about 3-20, about 4-19, about 5-18, about 6- 17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the second probe that is complementary to a portion of the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • each portion of the second probe is connected by an optional linker.
  • the optional linker is a nucleotide linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the second probe of the sets of probes used in the methods described herein comprises the structure:
  • ]-[ comprises an optional linker (e.g., a nucleotide linker).
  • ]-[ represents a direct linkage between two portions of the second probe (z.e., a phosphodiester bond).
  • the first probe of the sets of probes used in the methods described herein includes an oligonucleotide portion that is complementary to the second probe.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' end and the 3' end of the first probe.
  • the first probe of the sets of probes used in the methods for profiling interactions between RNA-binding proteins and RNAs disclosed herein also comprises an oligonucleotide portion that is complementary to an RNA of interest.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.
  • the first probe of the sets of probes used in the methods disclosed herein also comprises an oligonucleotide barcode sequence made up of a specific sequence of nucleotides.
  • the oligonucleotide barcode sequence of the first probe is about 1-10, about 2-9, about 3-8, about 4-7, or about 5-6 nucleotides in length.
  • the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.
  • the barcodes of the oligonucleotide probes described herein may comprise genespecific sequences used to identify RNAs of interest (z.e., RNAs that are bound by at least one RNA-binding protein).
  • the arrangement of the portions of the first oligonucleotide probe in any order is contemplated by the present disclosure.
  • a portion of the first probe is connected by an optional linker to another portion.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the first probe comprises the structure:
  • ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • any of the oligonucleotide portions of the probes that make up the sets of probes provided herein comprise DNA.
  • the third probe of the sets of probes used in the methods for profiling interactions between RNA-binding proteins and RNAs provided herein comprises a portion complementary to an RNA of interest and a portion complementary to the first probe of the set of probes.
  • the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.
  • the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the portion of the third probe that is complementary to a portion of the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
  • the third probe of the set of probes comprises the structure: 5'-[portion complementary to RNA of interest] -[portion complementary to first probe] -3', wherein ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • the use of any type of cell in the methods disclosed herein is contemplated by the present disclosure (e.g., any of the cell types described herein).
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the present disclosure also contemplates performing the methods for profiling epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs described herein on multiple cells simultaneously.
  • the method is performed on multiple cells of the same cell type.
  • the method is performed on multiple cells comprising cells of different cell types.
  • epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs are profiled in more than 10 cells, more than 20 cells, more than 50 cells, more than 100 cells, more than 200 cells, more than 300 cells, more than 400 cells, more than 500 cells, or more than 1000 cells simultaneously.
  • RNA-binding proteins and RNAs include, but are not limited to, stem cells, progenitor cells, neuronal cells, astrocytes, dendritic cells, endothelial cells, microglia, oligodendrocytes, muscle cells, myocardial cells, mesenchymal cells, epithelial cells, immune cells, hepatic cells, smooth and skeletal muscle cells, hematopoietic cells, lymphocytes, monocytes, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, and neurons.
  • the cell or cells are present within an intact tissue (e.g., of any of the tissue types described herein).
  • the intact tissue is a fixed tissue sample.
  • the intact tissue comprises multiple cell types.
  • the tissue is epithelial tissue, connective tissue, muscular tissue, or nervous tissue.
  • the tissue is cardiac tissue, lymph node tissue, liver tissue, muscle tissue, bone tissue, eye tissue, brain tissue, or ear tissue.
  • RNAs of interest for which epitranscriptomic modification or interactions with RNA-binding proteins are profiled in the methods described herein may be transcripts that have been expressed from the genomic DNA of the cell.
  • the RNAs of interest are messenger RNA (mRNA), transfer RNA (tRNA), and/or ribosomal RNA (rRNA).
  • the RNAs of interest comprise transcripts that have not been processed yet (e.g., pre-mRNA).
  • RNA-binding protein may be used to profile one epitranscriptomically-modified RNA or RNA bound by an RNA-binding protein in a cell at a time, or multiple epitranscriptomically-modified RNAs of interest or RNAs of interest bound by an RNA-binding protein simultaneously.
  • epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs in a cell, or in multiple cells are profiled for more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 RNAs simultaneously.
  • a polymeric matrix is used in the methods described herein following rolling circle amplification to facilitate sequencing and imaging of the epitranscriptomically-modified RNAs of interest or RNAs of interest bound by an RNA-binding protein in the cell.
  • the use of various polymeric matrices is contemplated by the present disclosure, and any polymeric matrix in which the one or more concatenated amplicons can be embedded is suitable for use in the methods described herein.
  • the polymeric matrix is a hydrogel (z.e., a network of crosslinked polymers that are hydrophilic).
  • the hydrogel is a polyvinyl alcohol hydrogel, a polyethylene glycol hydrogel, a polyacrylate hydrogel, or a polyacrylamide hydrogel.
  • the hydrogel is a polyacrylamide hydrogel.
  • a hydrogel may be prepared, for example, by incubating the sample in a buffer comprising acrylamide and bis-acrylamide, removing the buffer, and incubating the sample in a polymerization mixture (comprising, e.g., ammonium persulfate and tetramethylethylenediamine).
  • a polymerization mixture comprising, e.g., ammonium persulfate and tetramethylethylenediamine.
  • Such reagents may also be provided in a kit, e.g., a kit for performing any of the methods described herein, or any of the kits described herein.
  • the step of performing rolling circle amplification to amplify the circular oligonucleotide to produce one or more concatenated amplicons further comprises providing nucleotides modified with reactive chemical groups (e.g., amine modified nucleotides such as 5-(3-aminoallyl)-dUTP).
  • nucleotides modified with reactive chemical groups make up about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the nucleotides used in the amplification reaction.
  • the step of performing rolling circle amplification to amplify the circular oligonucleotide to produce one or more concatenated amplicons may further comprise providing amine- modified nucleotides such as 5-(3-aminoallyl)-dUTP.
  • the amine-modified nucleotides are incorporated into the one or more concatenated amplicons as they are produced.
  • the resulting amplicons are functionalized with primary amines, which can be further reacted with another compatible chemical moiety (e.g., A-hydroxysuccinimide) to facilitate the step of embedding the concatenated amplicons in the polymeric matrix.
  • another compatible chemical moiety e.g., A-hydroxysuccinimide
  • the step of embedding the one or more concatenated amplicons in a polymeric matrix comprises reacting the amine-modified nucleotides of the one or more concatenated amplicons with acrylic acid A-hydroxy succinimide ester and co-polymerizing the one or more concatenated amplicons and the polymer matrix.
  • the methods disclosed herein also include a step of sequencing the concatenated amplicons, or a portion thereof, embedded in the polymeric matrix.
  • the step of sequencing comprises performing “sequencing with error-reduction by dynamic annealing and ligation” (SEDAL sequencing). SEDAL sequencing is described further in Wang, X.
  • an oligonucleotide probe comprising a detectable label (z.e., any label that can be used to visualize the location of the additional oligonucleotide probe, for example, through imaging) is provided to the cell.
  • the detectable label is fluorescent (e.g., a fluorophore).
  • the additional oligonucleotide probe is complementary to the oligonucleotide barcode sequence of the first probe and is thus linked to the identity of an RNA of interest and can be used to identify the location of an RNA of interest within the cell (or within an organelle when the method is performed at subcellular resolution, e.g., with staining to identify the locations of individual organelles).
  • the additional oligonucleotide probe used in the methods described herein may be read out using any suitable imaging technique known in the art.
  • the additional oligonucleotide probe comprises a fluorophore
  • the fluorophore may be read out using imaging to identify the RNA of interest.
  • the additional oligonucleotide probe comprises a sequence complementary to a barcode sequence on the first oligonucleotide probe, which is used to detect a specific RNA of interest.
  • the step of imaging comprises fluorescent imaging. In certain embodiments, the step of imaging comprises confocal microscopy. In certain embodiments, the step of imaging comprises epifluorescence microscopy. In certain embodiments, two rounds of imaging are performed. In certain embodiments, three rounds of imaging are performed. In certain embodiments, four rounds of imaging are performed. In certain embodiments, five or more rounds of imaging are performed.
  • the methods for profiling epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs described herein may be combined with methods for profiling additional molecules within the cell.
  • additional RNAs including RNAs that have not been epitranscriptomically modified and RNAs that are not bound by an RNA-binding protein
  • expression of additional RNAs may be profiled alongside epitranscriptomically modified RNAs or RNAs bound by one or more RNA- binding proteins using probes that do not comprise a portion that recognizes an epitranscriptomic RNA modification or RNA-binding protein.
  • Methods for profiling other types of molecules e.g., DNAs, proteins, carbohydrates, or lipids
  • DNAs, proteins, carbohydrates, or lipids may be combined with the methods described herein as well.
  • profiling unmodified RNAs comprises: a) contacting the cell with one or more pairs of probes, wherein each pair of probes comprises a first probe and a second probe, wherein: i) the first probe comprises an oligonucleotide portion complementary to a portion of the second probe, an oligonucleotide barcode sequence, and an oligonucleotide portion complementary to an unmodified RNA of interest; and ii) the second probe comprises a portion that is complementary to the unmodified RNA of interest and a portion that is complementary to a portion of the first probe; b) ligating the 5' end and the 3' end of the first probe together to produce a circular oligonucleotide; c) performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons; d
  • the methods provided herein further comprise determining the cell type of the profiled cell by comparing the epitranscriptomic RNA modification profile (or profile of interactions between RNAs and RNA-binding proteins) of a cell to reference data comprising epitranscriptomic RNA modification profiles (or profiles of interactions between RNAs and RNA-binding proteins) of various cell types.
  • the method further comprises overexpressing or knocking out one or more genes in the cell to determine whether the one or more genes are involved in epitranscriptomic modification of the RNA of interest.
  • the method further comprises repeating steps (a)- (e) at multiple time points to profile epitranscriptomic RNA modification or interactions between RNA-binding proteins and RNA in the cell over time.
  • the methods further comprise examining how the profile of epitranscriptomically modified RNAs or interactions between RNAs and RNA-binding proteins in a cell or multiple cells is affected by an immune response within an intact tissue, or how the profile is affected due to proximity to a tumor.
  • any of the methods described herein further comprise resolving the locations of the modified RNAs of interest at subcellular resolution (/'. ⁇ ?., within specific organelles within the cells; approximately 150-400 nm spatial resolution, depending on both the size of DNA amplicons and optical limits).
  • This may be accomplished through organelle staining procedures, which are well known in the art.
  • various organelles such as the endoplasmic reticulum (ER), the cytoskeleton, and the mitochondria may be stained.
  • the agents used to stain various organelles in the cells comprise small molecule dyes, antibodies, and/or protein dyes.
  • the present disclosure provides methods for diagnosing a disease or disorder in a subject.
  • the methods for profiling epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs described herein may be performed on a cell or multiple cells (e.g., in an intact tissue) taken from a subject (e.g., a subject who is thought to have or is at risk of having a disease or disorder, or a subject who is healthy or thought to be healthy).
  • RNAs of interest in the cell can then be compared to the expression of the same RNAs of interest in a non-diseased cell or a cell from a non-diseased tissue sample (e.g., a cell from a healthy individual, or multiple cells from a population of healthy individuals). Any difference in the epitranscriptomic RNA modification profile or the profile of interactions between RNA-binding proteins and RNAs of the cell (including of a single RNA or of multiple RNAs of interest, e.g., a specific disease signature) relative to one or more non-diseased cells may indicate that the subject has the disease or disorder.
  • Epitranscriptomic RNA modifications or interactions between RNA- binding proteins and RNAs in one or more non-diseased cells may be profiled alongside expression in a diseased cell as a control experiment.
  • Epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs in one or more non-diseased cells may have also been profiled previously, and expression in a diseased cell may be compared to this reference data for a non-diseased cell.
  • a method for diagnosing a disease or disorder in a subject comprises the steps of: a) contacting a cell taken from a subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion
  • a method for diagnosing a disease or disorder in a subject comprises the steps of: a) contacting a cell (or in multiple cells, e.g., in an intact tissue) taken from a subject with one or more pairs of probes, wherein each pair of probes comprises a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b) lig
  • a method for diagnosing a disease or disorder in a subject comprises the steps of: a) contacting a cell taken from a subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe; and iii
  • epitranscriptomic RNA modifications in one or more nondiseased cells, or the interactions between RNAs and RNA-binding proteins in one or more non-diseased cells are profiled simultaneously alongside the cell taken from a subject using the methods disclosed herein as a control experiment.
  • the profile of epitranscriptomic RNA modifications in one or more non-diseased cells, or the profile of interactions between RNAs and RNA-binding proteins in one or more non-diseased cells, that is compared to expression in a diseased cell comprises reference data from when the method was performed on one or more non-diseased cells previously.
  • the epitranscriptomic RNA modification is N 6 -methyladenosine (m 6 A), N’-mcthyladcnosinc (m 1 A), pseudouridine, N 6 ,2'-O-dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -acetylcytidine (ac 4 C), 2'- O-methylation (Nm), or 5-methylcytosine (m 5 C).
  • Other epitranscriptomic modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology.
  • the epitranscriptomic RNA modification is an adenosine-to-inosine modification.
  • the epitranscriptomic modification is queuosine (i.e., queuosine replaces another nucleotide in the RNA), polyadenylation, intron splicing, or histone mRNA processing.
  • the epitranscriptomic RNA modification is N 6 - methyladenosine (m 6 A).
  • a single epitranscriptomic modification may be profiled in a cell to diagnose a disease or disorder in a subject using the methods disclosed herein, or multiple different epitranscriptomic modifications (e.g., two, three, four, five, or more) may be profiled in the cell simultaneously.
  • Profiling of interactions between RNAs and any RNA- binding protein is also contemplated by the present disclosure.
  • the RNA-binding protein is an enzyme that introduces an epitranscriptomic modification onto an RNA.
  • the RNA-binding protein is a YTH family protein (e.g., YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2), an IGF2BP family protein (e.g., IGF2BP1, IGF2BP2, or IGF2BP3), or FMRI.
  • YTH family protein e.g., YTHDF1, YTHDF2, YTHDF3, YTHDC1, or YTHDC2
  • IGF2BP family protein e.g., IGF2BP1, IGF2BP2, or IGF2BP3
  • FMRI FMRI
  • Interactions between RNAs and a single RNA-binding protein may be profiled in a cell to diagnose a disease or disorder in a subject using the methods disclosed herein, or interactions with multiple different RNA-binding proteins (e.g., two, three, four, five, or more) may be profiled in the cell simultaneously.
  • Diagnosis of any disease or disorder is contemplated
  • the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease.
  • GI gastrointestinal
  • the cell is present in a tissue (e.g., epithelial tissue, connective tissue, muscular tissue, or nervous tissue).
  • the tissue is a tissue sample from a subject.
  • the subject is a non-human experimental animal (e.g., a mouse).
  • the subject is a domesticated animal.
  • the subject is a human.
  • the tissue sample comprises a fixed tissue sample.
  • the tissue sample is a biopsy (e.g., bone, bone marrow, breast, gastrointestinal tract, lung, liver, pancreas, prostate, brain, nerve, renal, endometrial, cervical, lymph node, muscle, or skin biopsy).
  • the biopsy is a tumor biopsy.
  • the tissue is brain tissue.
  • the tissue is from the central nervous system.
  • the present disclosure provides methods for treating a disease or disorder in a subject.
  • the methods for profiling epitranscriptomic RNA modification or interactions between RNA-binding proteins and RNAs described herein may be performed in a cell (or in multiple cells, e.g., in an intact tissue) from a sample taken from a subject (e.g., a subject who is thought to have or is at risk of having a disease or disorder).
  • the profile of epitranscriptomic modifications of one or more RNAs or the profile of interactions between RNA-binding proteins and one or more RNAs in the cell can then be compared to the epitranscriptomic modification profile or the profile of interactions with RNA-binding proteins of the same RNAs of interest in a cell from a non-diseased tissue sample.
  • a treatment for the disease or disorder may then be administered to the subject if any difference in the profile of epitranscriptomic RNA modifications or the profile of interactions between RNA-binding proteins and one or more RNAs in the cell relative to a non-diseased cell is observed.
  • Epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in one or more non-diseased cells may be profiled alongside epitranscriptomic RNA modification in a diseased cell as a control experiment.
  • Epitranscriptomic RNA modification and/or interactions between RNA-binding proteins and RNAs in one or more non-diseased cells may have also been profiled previously, and the profile of epitranscriptomic RNA modification or interactions between RNA-binding proteins and RNAs in a diseased cell may be compared to this reference data for a non-diseased cell.
  • the present disclosure provides methods for treating a disease or disorder in a subject comprising the steps of: a) contacting a cell taken from a subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe; and
  • the present disclosure provides methods for treating a disease or disorder in a subject comprising the steps of: a) contacting a cell (or in multiple cells, e.g., in an intact tissue) taken from the subject with one or more pairs of probes, wherein each pair of probes comprises a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; b)
  • the present disclosure provides methods for treating a disease or disorder in a subject comprising the steps of: a) contacting a cell taken from a subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion
  • epitranscriptomic RNA modification in one or more nondiseased cells, or the interactions between RNAs and RNA-binding proteins in one or more non-diseased cells is profiled simultaneously using the methods disclosed herein as a control experiment.
  • the epitranscriptomic RNA modification data in one or more non-diseased cells, or the profile of interactions between RNAs and RNA-binding proteins in one or more non-diseased cells, that is compared to the profile of a diseased cell comprises reference data from a time the method was performed on a non-diseased cell previously.
  • any suitable treatment for a disease or disorder may be administered to the subject.
  • the treatment comprises administering a therapeutic agent.
  • the treatment comprises surgery.
  • the treatment comprises imaging.
  • the treatment comprises performing further diagnostic methods.
  • the treatment comprises radiation therapy.
  • the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate.
  • the therapeutic agent is a known drug and/or an FDA-approved drug.
  • the protein is an antibody.
  • the protein is an antibody variant.
  • the protein is a receptor, or a fragment or variant thereof.
  • the protein is a cytokine.
  • the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).
  • the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, a neurological disorder, an ophthalmic disease, or a cardiovascular disease.
  • GI gastrointestinal
  • CNS central nervous system
  • the subject is a human.
  • the sample comprises a biological sample.
  • the sample comprises a tissue sample.
  • the tissue sample is a biopsy (e.g., bone, bone marrow, breast, gastrointestinal tract, lung, liver, pancreas, prostate, brain, nerve, renal, endometrial, cervical, lymph node, muscle, or skin biopsy).
  • the biopsy is a tumor biopsy.
  • the biopsy is a solid tumor biopsy.
  • the tissue sample is a brain tissue sample.
  • the tissue sample is a central nervous system tissue sample.
  • the epitranscriptomic profile of the biological sample informs prognostic decisions that guide therapies including but not limited to, pharmacological interventions for treating various conditions such as diabetes, psychiatric disorders, liver disease, kidney disease, blood disease, endocrine or exocrine disorders, heart disease, cancer therapies such as chemotherapy, targeted therapies, immunotherapy (e.g., checkpoint inhibition, CAR-T, cancer vaccines, etc.), metabolic disorders, or immune and autoimmune disorders.
  • guide therapies including but not limited to, pharmacological interventions for treating various conditions such as diabetes, psychiatric disorders, liver disease, kidney disease, blood disease, endocrine or exocrine disorders, heart disease, cancer therapies such as chemotherapy, targeted therapies, immunotherapy (e.g., checkpoint inhibition, CAR-T, cancer vaccines, etc.), metabolic disorders, or immune and autoimmune disorders.
  • the present disclosure provides methods for screening for an agent capable of modulating epitranscriptomic modification of one or more RNAs of interest or modulating interactions between RNA-binding proteins and RNAs or one or more RNAs of interest.
  • the methods for profiling epitranscriptomic RNA modifications or interactions between RNA-binding proteins and RNAs described herein may be performed in a cell (or in multiple cells, e.g., in an intact tissue) in the presence of one or more candidate agents.
  • RNA-binding protein in the cell (e.g., a normal cell, or a diseased cell) can then be compared to the expression of the same RNAs of interest in a cell that was not exposed to the one or more candidate agents. Any difference in the profile of epitranscriptomic RNA modification or the profile of interactions between RNA-binding proteins and RNAs relative to the cell that was not exposed to the candidate agent(s) may indicate that epitranscriptomic modification of the one or more RNAs of interest, or the interactions between the one or more RNAs of interest and one or more RNA-binding proteins, is modulated by the candidate agent(s).
  • a particular signature (e.g., of epitranscriptomic modification of multiple RNAs of interest, or of the interaction of an RNA-binding protein with multiple RNAs of interest) that is known to be associated with treatment of a disease may be used to identify a candidate agent capable of modulating epitranscriptomic RNA modification in a desired manner.
  • the methods described herein may also be used to identify drugs that have certain side effects, for example, by looking for specific epitranscriptomic RNA modification signatures or signatures of the interactions between an RNA-binding protein and RNAs of interest when one or more cells is treated with a candidate agent or known drug.
  • the present disclosure provides methods for screening for an agent capable of modulating epitranscriptomic modification of one or more RNAs comprising the steps of: a) contacting a cell that is being treated with or has been treated with a candidate agent with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the
  • the present disclosure provides methods for screening for an agent capable of modulating epitranscriptomic modification of one or more RNAs comprising the steps of: a) contacting a cell (or in multiple cells, e.g., in an intact tissue) that is being treated with or has been treated with a candidate agent (or combinations of multiple candidate agents and/or known drugs, e.g., as provided in a screening library of compounds) with one or more pairs of probes, wherein each pair of probes comprises a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and
  • the present disclosure provides methods for screening for an agent capable of modulating interactions between RNA and RNA-binding proteins, the method comprising: a) contacting a cell taken from a subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, and a third probe wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleot
  • the candidate agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate.
  • the candidate agent comprises a known drug or an FDA-approved drug.
  • the protein is an antibody.
  • the protein is an antibody fragment or an antibody variant.
  • the protein is a receptor.
  • the protein is a cytokine.
  • the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).
  • multiple candidate agents are provided as a screening library. Any candidate agent may be screened using the methods described herein. In particular, any candidate agents thought to be capable of modulating epitranscriptomic modification of one or more RNAs of interest or interaction of one or more RNAs of interest with an RNA-binding protein may be screened using the methods described herein.
  • modulation of epitranscriptomic modification of one or more RNAs of interest and/or interaction of one or more RNAs of interest with an RNA-binding protein by the candidate agent is associated with reducing, relieving, or eliminating the symptoms of a disease or disorder, or preventing the development or progression of the disease or disorder.
  • the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease.
  • GI gastrointestinal
  • the present disclosure also provides pairs of probes for use in the methods and systems for profiling epitranscriptomic RNA modifications described herein.
  • the present disclosure provides pairs of probes comprising a first probe (also referred to herein as the “padlock” probe) and a second probe (also referred to herein as the “primer” probe), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe.
  • the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleot
  • All of the probes described herein may optionally have spacers or linkers of various nucleotide lengths in between each of the recited components, or the components of the oligonucleotide probes may be joined directly to one another (z.e., by a phosphodiester bond). All of the probes described herein may comprise standard nucleotides, or some of the standard nucleotides may be substituted for any modified nucleotides known in the art.
  • the second probe of the pair of probes (also referred to herein as the “primer probe”) comprises a portion that recognizes an epitranscriptomic RNA modification (/'. ⁇ ?., a post- transcriptional modification present on a particular RNA of interest).
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a peptide. In some embodiments, the portion of the second probe that binds the epitranscriptomic RNA modification comprises an aptamer. In some embodiments, the portion of the second probe that binds the epitranscriptomic RNA modification comprises a small molecule. In certain embodiments, the second probe binds to the epitranscriptomic modification through a mechanism comprising a biotin-streptavidin interaction.
  • the portion of the probe that recognizes an epitranscriptomic RNA modification may be a protein (e.g., an antibody or antibody variant, or any protein that is otherwise capable of binding to a specific epitranscriptomic modification).
  • the protein is PAPG.
  • PAPG recognizes and binds an antibody that recognizes an epitranscriptomic RNA modification.
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an agent that binds an antibody, or an antibody variant.
  • the second probe may comprise a secondary antibody, and a primary antibody may be used to bind to the epitranscriptomic RNA modification. The secondary antibody on the second probe then recognizes the primary antibody bound to the epitranscriptomic RNA modification. See, for example, FIG. 1.
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant.
  • the secondary antibody may bind a primary antibody that recognizes the epitranscriptomic RNA modification.
  • the primary antibody is an anti-m 6 A antibody, an anti-m 1 A antibody, an anti-pseudouridine antibody, an anti-m 6 Am antibody, an anti-m 7 G antibody, an anti-ac 4 C antibody, an anti-Nm antibody, or an anti-m 5 C antibody.
  • Other epitranscriptomic modifications include those described in Kumar, S. et al., Frontiers in Cell and Developmental Biology. 9 (2021); and Harcourt, E. M. et al., Nature.
  • the present disclosure also contemplates the use of any agent capable of binding an epitranscriptomic RNA modification on the probes described herein.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a protein.
  • the protein is an m 6 A-specific YTH domain protein, or a portion thereof.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises an antibody, or an antibody variant.
  • the second probe also comprises a portion that is complementary to a portion of the first probe.
  • the portions of the first and second probes that are complementary to one another may be the same on each set of probes. In some embodiments, the portions of the first and second probes that are complementary to one another are unique on each pair of probes. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is about 3-20, about 4-19, about 5- 18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the second probe that is complementary to a portion of the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • each portion of the second probe is connected by an optional linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the linker is a non-nucleotide linker (e.g.. amino acids, chemical linkers, polymers, etc.).
  • the second probe of the pair of probes comprises the structure:
  • ]-[ comprises an optional linker (e.g.. a nucleotide linker).
  • ]-[ represents a direct linkage between two portions of the second probe (z.e., a phosphodiester bond).
  • the first probe of the pair of probes provided herein includes an oligonucleotide portion that is complementary to the second probe.
  • the portion of the first probe that is complementary to a portion of the second probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the first probe that is complementary to a portion of the second probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' end and the 3' end of the first probe.
  • the first probe of the pair of probes provided herein also comprises an oligonucleotide portion that is complementary to an RNA of interest.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.
  • the first probe of the pair of probes disclosed herein also comprises an oligonucleotide barcode sequence made up of a specific sequence of nucleotides.
  • the oligonucleotide barcode sequence of the first probe is about 1-10, about 2- 9, about 3-8, about 4-7, or about 5-6 nucleotides in length.
  • the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.
  • the barcodes of the oligonucleotide probes described herein may comprise gene-specific sequences used to identify RNAs of interest (z.e., RNAs that have been modified with at least one epitranscriptomic modification).
  • each portion of the first probe is connected by an optional linker.
  • the optional linker is a nucleotide linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the optional linkers comprise other types of linkages besides nucleotides (e.g.. chemical linkers, peptide linkers, etc.).
  • the first probe comprises the structure:
  • ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • any of the oligonucleotide portions of the first probe and/or the second probe comprise DNA.
  • the present disclosure also provides sets of probes for use in the methods and systems for profiling epitranscriptomic RNA modifications described herein.
  • the present disclosure provides pairs of probes comprising a first probe (also referred to herein as the “padlock” probe) and a second probe (also referred to herein as the “splint” probe), and a third probe (also referred to herein as the “primer” probe), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an
  • All of the probes in the sets of probes described herein may optionally have spacers or linkers of various nucleotide lengths in between each of the recited components, or the components of the oligonucleotide probes may be joined directly to one another (z.e., by a phosphodiester bond). All of the probes described herein may comprise standard nucleotides, or some of the standard nucleotides may be substituted for any modified nucleotides known in the art.
  • the second probe of the set of probes (also referred to herein as the “splint probe”) comprises a portion that recognizes an epitranscriptomic RNA modification (z.e., a post- transcriptional modification present on a particular RNA of interest).
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a peptide.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises an aptamer.
  • the portion of the second probe that binds the epitranscriptomic RNA modification comprises a small molecule.
  • the second probe binds to the epitranscriptomic modification through a mechanism comprising a biotin-streptavidin interaction.
  • the portion of the probe that recognizes an epitranscriptomic RNA modification may be a protein (e.g., an antibody or antibody variant, or any protein that is otherwise capable of binding to a specific epitranscriptomic modification).
  • the protein is PAPG.
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an agent that binds an antibody, or an antibody variant.
  • the second probe may comprise a secondary antibody, and a primary antibody may be used to bind to the epitranscriptomic RNA modification.
  • the second probe may comprise the protein PAPG, which may bind an antibody that binds to the epitranscriptomic RNA modification.
  • PAPG recognizes and binds to an antibody
  • the antibody recognizes and binds to the epitranscriptomic RNA modification.
  • the portion of the second probe that recognizes the epitranscriptomic RNA modification comprises an antibody (e.g., a secondary antibody), or an antibody variant.
  • the secondary antibody recognizes and binds to a primary antibody that recognizes and binds to the epitranscriptomic RNA.
  • the cell may be contacted with the primary antibody before or after being contacted with the one or more pairs of probes. In certain embodiments, the cell is contacted with the primary antibody prior to being contacted with the one or more pairs of probes.
  • the primary antibody is an anti- m 6 A antibody, an anti-m 1 A antibody, an anti-pseudouridine antibody, an anti-m 6 Am antibody, an anti-m 7 G antibody, an anti-ac 4 C antibody, an anti-Nm antibody, or an anti-m 5 C antibody.
  • the present disclosure also contemplates the use of any agent capable of binding an epitranscriptomic RNA modification directly on the probes described herein.
  • the portion of the second probe that binds the epitranscriptomic RNA modification directly comprises a protein.
  • the protein is an m 6 A-specific YTH domain protein.
  • the portion of the second probe that binds the epitranscriptomic RNA modification directly comprises an antibody, or an antibody variant.
  • the second probe of the set of probes further comprises a polymerization blocker.
  • the polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the methods described herein.
  • the polymerization blocker is at the 3' end of the second oligonucleotide probe.
  • the polymerization blocker can be, for example, any chemical moiety that prevents a polymerase from using the second oligonucleotide probe as a primer for polymerization.
  • the polymerization blocker is a nucleic acid residue comprising a blocked 3' hydroxyl group (e.g., comprising an oxygen protecting group on the 3' hydroxyl group).
  • the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group.
  • the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added.
  • the polymerization blocker comprises an inverted nucleic acid residue.
  • the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue.
  • the polymerization blocker is an inverted thymine residue.
  • the second probe also comprises a portion that is complementary to a portion of the first probe.
  • the portions of the first and second probes that are complementary to one another may be the same on each set of probes. In some embodiments, the portions of the first and second probes that are complementary to one another are unique on each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is about 3-20, about 4-19, about 5-18, about 6-17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the second probe that is complementary to a portion of the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • each portion of the second probe is connected by an optional linker.
  • the optional linker is a nucleotide linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the second probe of the sets of probes described herein comprises the structure:
  • ]-[ comprises an optional linker (e.g., a nucleotide linker).
  • ]-[ represents a direct linkage between two portions of the second probe (z.e., a phosphodiester bond).
  • the first probe of the sets of probes described herein includes an oligonucleotide portion that is complementary to the second probe.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' end and the 3' end of the first probe.
  • the first probe of the sets of probes disclosed herein also comprises an oligonucleotide portion that is complementary to an RNA of interest.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.
  • the first probe of the sets of probes disclosed herein also comprises an oligonucleotide barcode sequence made up of a specific sequence of nucleotides.
  • the oligonucleotide barcode sequence of the first probe is about 1-10, about 2- 9, about 3-8, about 4-7, or about 5-6 nucleotides in length.
  • the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.
  • the barcodes of the oligonucleotide probes described herein may comprise gene-specific sequences used to identify RNAs of interest (z.e., RNAs that have been modified with at least one particular epitranscriptomic modification).
  • the arrangement of the portions of the first oligonucleotide probe in any order is contemplated by the present disclosure.
  • a portion of the first probe is connected by an optional linker to another portion.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the first probe comprises the structure:
  • ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • any of the oligonucleotide portions of the probes that make up the sets of probes provided herein comprise DNA.
  • the third probe of the sets of probes provided herein comprises a portion complementary to an RNA of interest and a portion complementary to the first probe of the set of probes.
  • the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.
  • the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the portion of the third probe that is complementary to a portion of the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
  • the third probe of the set of probes comprises the structure: [0143] 5'-[portion complementary to RNA of interest] -[portion complementary to first probe] -3', wherein ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • the present disclosure also provides sets of probes for use in the methods and systems for profiling interactions between RNA-binding proteins and RNAs described herein.
  • the present disclosure provides pairs of probes comprising a first probe (also referred to herein as the “padlock” probe) and a second probe (also referred to herein as the “splint” probe), and a third probe (also referred to herein as the “primer” probe), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises
  • the second probe of the set of probes for profiling interactions between RNA-binding proteins and RNAs comprises a portion that recognizes an RNA-binding protein (e.g., an enzyme that introduces an epitranscriptomic modification onto an RNA).
  • the portion of the second probe that binds the RNA-binding protein comprises a peptide.
  • the portion of the second probe that binds the RNA-binding protein comprises an aptamer.
  • the portion of the second probe that binds the RNA-binding protein comprises a small molecule.
  • the second probe binds to the RNA-binding protein through a mechanism comprising a biotin-streptavidin interaction.
  • the portion of the probe that recognizes an RNA-binding protein may be a protein (e.g., an antibody or antibody variant, or any protein that is otherwise capable of binding to a specific RNA-binding protein).
  • the portion of the second probe that recognizes the RNA- binding protein comprises an agent that binds an antibody, or an antibody variant.
  • the second probe may comprise a secondary antibody, and a primary antibody may be used to bind to the RNA-binding protein. The secondary antibody on the second probe then recognizes the primary antibody bound to the RNA-binding protein. See, for example, FIGs.
  • the portion of the second probe that recognizes the RNA-binding protein comprises an antibody (e.g., a secondary antibody), or an antibody variant.
  • the secondary antibody recognizes and binds to a primary antibody that recognizes the RNA-binding protein.
  • the cell may be contacted with the primary antibody before or after being contacted with the one or more pairs of probes. In certain embodiments, the cell is contacted with the primary antibody prior to being contacted with the one or more pairs of probes. In place of a primary antibody, the present disclosure also contemplates the use of any agent capable of recognizing and directly binding an RNA- binding protein on the probes described herein.
  • the portion of the second probe that binds the RNA-binding protein directly comprises a protein. In some embodiments, the portion of the second probe that binds the RNA-binding protein directly comprises an antibody, or an antibody variant.
  • the second probe of the set of probes further comprises a polymerization blocker.
  • the polymerization blocker can be any moiety capable of preventing the use of the second oligonucleotide probe as a primer in the rolling circle amplification of step (c) of the methods described herein. In some embodiments, the polymerization blocker is at the 3' end of the second oligonucleotide probe.
  • the polymerization blocker can be, for example, any chemical moiety that prevents a polymerase from using the second oligonucleotide probe as a primer for polymerization.
  • the polymerization blocker is a nucleic acid residue comprising a blocked 3' hydroxyl group (e.g., comprising an oxygen protecting group on the 3' hydroxyl group).
  • the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group.
  • the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added.
  • the polymerization blocker comprises an inverted nucleic acid residue.
  • the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.
  • the second probe also comprises a portion that is complementary to a portion of the first probe.
  • the portions of the first and second probes that are complementary to one another may be the same on each set of probes. In some embodiments, the portions of the first and second probes that are complementary to one another are unique on each of the first and second probes. In some embodiments, the portion of the second probe that is complementary to a portion of the first probe is about 3-20, about 4-19, about 5-18, about 6- 17, about 7-16, about 8-15, about 9-14, about 10-13, or about 11-12 nucleotides in length.
  • the portion of the second probe that is complementary to a portion of the first probe is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • each portion of the second probe is connected by an optional linker.
  • the optional linker is a nucleotide linker.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the second probe of the sets of probes comprises the structure:
  • ]-[ comprises an optional linker (e.g., a nucleotide linker).
  • ]-[ represents a direct linkage between two portions of the second probe (z.e., a phosphodiester bond).
  • the first probe of the sets of probes used in the methods for profiling interactions between RNA-binding proteins and RNAs described herein includes an oligonucleotide portion that is complementary to the second probe.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4-20, about 5-19, about 6-18, about 7-17, about 8-16, about 9-15, about 10-14, or about 11-13 nucleotides in length.
  • the portion of the first probe that is complementary to a portion of the second probe is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the second probe is split between the 5' end and the 3' end of the first probe.
  • the first probe of the sets of probes used in the methods for profiling interactions between RNA-binding proteins and RNAs disclosed herein also comprises an oligonucleotide portion that is complementary to an RNA of interest.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10-30, about 11-29, about 12-28, about 13-27, about 14-26, about 15-25, about 16-24, about 17-23, about 18-22, or about 19-21 nucleotides in length.
  • the oligonucleotide portion of the first probe that is complementary to the RNA of interest is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more than 30 nucleotides in length.
  • the first probe of the sets of probes disclosed herein also comprises an oligonucleotide barcode sequence made up of a specific sequence of nucleotides.
  • the oligonucleotide barcode sequence of the first probe is about 1-10, about 2- 9, about 3-8, about 4-7, or about 5-6 nucleotides in length.
  • the oligonucleotide barcode sequence of the first probe is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.
  • the barcodes of the oligonucleotide probes described herein may comprise gene-specific sequences used to identify RNAs of interest (z.e., RNAs that are bound by at least one RNA-binding protein).
  • RNAs of interest z.e., RNAs that are bound by at least one RNA-binding protein.
  • the arrangement of the portions of the first oligonucleotide probe in any order is contemplated by the present disclosure.
  • a portion of the first probe is connected by an optional linker to another portion.
  • the optional linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long.
  • the first probe comprises the structure:
  • ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • any of the oligonucleotide portions of the probes that make up the sets of probes provided herein comprise DNA.
  • the third probe of the sets of probes used in the methods for profiling interactions between RNA-binding proteins and RNAs provided herein comprises a portion complementary to an RNA of interest and a portion complementary to the first probe of the set of probes.
  • the portion of the third probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.
  • the portion of the third probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the portion of the third probe that is complementary to a portion of the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
  • the third probe of the set of probes comprises the structure: 5'-[portion complementary to RNA of interest] -[portion complementary to first probe] -3', wherein ]-[ comprises an optional nucleotide linker.
  • ]-[ represents a direct linkage between two portions of the first probe (z.e., a phosphodiester bond).
  • All of the probes described herein may optionally have spacers or linkers of various nucleotide lengths in between each of the recited components, or the components of the oligonucleotide probes may be joined directly to one another (z.e., by a phosphodiester bond). All of the probes described herein may comprise standard nucleotides, or some of the standard nucleotides may be substituted for any modified nucleotides known in the art.
  • the present disclosure provides a plurality of probes comprising multiple pairs of probes or sets of probes as described herein.
  • each pair of probes or set of probes in the plurality of probes comprises oligonucleotide portions that are complementary to a different RNA of interest.
  • the plurality of probes comprises more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 pairs of probes or sets of probes.
  • the present disclosure provides libraries comprising multiple sets of any of the probes described herein. In some aspects, the present disclosure provides libraries comprising multiple pairs of any of the probes provided herein. In some aspects, the present disclosure provides libraries comprising multiple first probes from the sets or pairs of probes provided herein, multiple second probes from the sets or pairs of probes provided herein, or multiple third probes from the sets of probes provided herein.
  • kits may comprise one or more of the probes as described herein. In some embodiments, the kits comprise any of the pairs of probes or sets of probes described herein, or multiple pairs of probes or sets of probes. In certain embodiments, the kits comprise more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, or more than 3000 pairs of probes or sets of probes. In some embodiments, the kits may further comprise a container (e.g., a vial, ampule, bottle, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampule, bottle, and/or dispenser package, or other suitable container.
  • kits may also comprise cells for performing control experiments.
  • the kits may further comprise other reagents for performing the methods disclosed herein (e.g., enzymes such as a ligase or a polymerase, amine-modified nucleotides as described herein, primary antibodies, secondary antibodies, buffers, and/or reagents and monomers for making a polymeric matrix (e.g., a polyacrylamide matrix)).
  • enzymes such as a ligase or a polymerase
  • amine-modified nucleotides as described herein primary antibodies
  • secondary antibodies e.g., buffers, and/or reagents and monomers for making a polymeric matrix
  • the kits are useful for profiling epitranscriptomic RNA modifications in a cell.
  • kits are additionally useful for profiling unmodified RNAs alongside epitranscriptomically-modified RNAs (/'. ⁇ ?., the kits also include pairs of probes for profiling unmodified RNAs of interest). In some embodiments, the kits are useful for profiling interactions between RNA-binding proteins and RNAs in a cell. In some embodiments, the kits are useful for diagnosing a disease in a subject. In some embodiments, the kits are useful for screening for an agent capable of modulating epitranscriptomic modification of one or more RNAs. In some embodiments, the kits are useful for diagnosing a disease or disorder in a subject. In some embodiments, the kits are useful for treating a disease or disorder in a subject. In certain embodiments, a kit described herein further includes instructions for using the kit.
  • the present disclosure provides systems for profiling epitranscriptomic RNA modification in a cell.
  • a system comprises: a) a cell (or in multiple cells, e.g., in an intact tissue); b) one or more pairs of probes comprising a first probe (z.e., the “padlock probe”) and a second probe (z.e., the “primer probe”), wherein: i) the first probe comprises an oligonucleotide portion that is complementary to the second probe, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide barcode sequence (e.g., a unique sequence used to identify each RNA of interest, for example, by SEDAL sequencing as discussed herein); and ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope
  • the present disclosure provides systems comprising: a) a cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an epitranscriptomic RNA modification and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope; and d) a computer.
  • the first probe comprises an oligon
  • the present disclosure provides systems comprising: a) a cell; b) one or more sets of probes comprising a first probe, a second probe, and a third probe, wherein: i) the first probe comprises an oligonucleotide portion that is complementary to a portion of the second probe, an oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to an RNA of interest, and an oligonucleotide portion that is complementary to a portion of the third probe; ii) the second probe comprises a portion that recognizes an RNA-binding protein and an oligonucleotide portion that is complementary to a portion of the first probe; and iii) the third probe comprises an oligonucleotide portion that is complementary to the RNA of interest and an oligonucleotide portion that is complementary to a portion of the first probe; c) a microscope; and d) a computer.
  • the first probe comprises an oligonucle
  • the epitranscriptomic RNA modification is N 6 -methyladenosine (m 6 A), N’-mcthyladcnosinc (m 1 A), pseudouridine, N 6 ,2'-O-dimethyladenosine (m 6 Am), 7-methylguanosine (m 7 G), N 4 -acetylcytidine (ac 4 C), 2'- O-methylation (Nm), or 5-methylcytosine (m 5 C).
  • the epitranscriptomic RNA modification is N 6 -methyladenosine (m 6 A).
  • a system further comprises software running on the computer.
  • the systems may further comprise other reagents (e.g., enzymes such as a ligase or a polymerase, amine-modified nucleotides as described herein, primary antibodies, secondary antibodies, buffers, and/or reagents and monomers for making a polymeric matrix (e.g., a polyacrylamide matrix)).
  • enzymes such as a ligase or a polymerase, amine-modified nucleotides as described herein, primary antibodies, secondary antibodies, buffers, and/or reagents and monomers for making a polymeric matrix (e.g., a polyacrylamide matrix)).
  • Example 1 Single-cell profiling of the epitrans criptome in situ
  • RNA modification of the transcriptome plays a crucial role in the regulation of RNA activities, processing (e.g., of pre-mRNA), stability, export, and translation. These chemical modifications of cellular RNA are diverse and ubiquitous, providing an additional layer of complexity in regulating gene expression.
  • Epitranscriptomics has been explored in second-generation sequencing, but to advance understanding of RNA modification, there is need for a strategy to study spatial arrangements of modified RNAs in the native cellular environment, as well as in a multiplexed and high-throughput way.
  • the methods described herein address these two issues through the use of probes conjugating an RNA modificationbinding moiety (e.g., an RNA-modification binding antibody) with an oligonucleotide primer.
  • the identity of RNAs with specific epitranscriptomic modifications can thus be sequentially decoded and identified using oligonucleotide barcode sequences present on the probes (FIG.l).
  • thi s method The utility of thi s method is demonstrated through detection of one of the most abundant and prevalent RNA modifications, N6-methyladenosine (m 6 A), a crucial epitranscriptomic marker in mRNA processing, translation, and degradation.
  • m 6 A-modified beta-actin transcripts were detected in cells (FIGs. 2A-2D).
  • Beta-actin is a common and abundant mRNA in cells modified by m 6 A.
  • Raw images show that the signals corresponding to beta-actin mRNAs can be seen (FIG. 2A) in comparison to several negative controls (FIGs. 2B-2D).
  • RNA modifications are not just subtle structural modifiers, but also active gene regulators.
  • mRNA modifications e.g., N 6 - methyladenosine
  • mRNA modifications impact almost every step of an RNA transcript’s life cycle from birth to death, including splicing, nuclear export, storage, cellular localization, translation, and decay.
  • mRNA modifications are non- stoichiometric. For a given gene, many modification sites are not 100% marked, and a number of RNA-de- modifying enzymes dynamically tune the status of modifications.
  • mRNA modifications affect the subcellular locations of mRNAs inside cells. Given the non- stoichiometric nature of mRNA modifications, there are at least two plausible hypotheses: within a single cell, mRNAs with different modification status have different spatiotemporal properties in order to precisely control the location and duration of protein production; or the apparent non- stoichiometry is a result of mixing different cell states and cell types, as previous measurements were all conducted with millions of cells. Finally, it is also unknown how the effects of mRNA modification vary between different cell types and states.
  • RNA modifications can either only target one gene at a time, or detect all the modification sites as a sum without differentiating by gene identity. It is noteworthy that many modified RNAs are regulated as large gene groups. Thus, it is necessary to simultaneously measure at least hundreds of modified genes (ideally transcriptome-wide) for meaningful data analysis.
  • the methods described herein represents a transformative toolbox for singlecell epitranscriptomic profiling with spatial resolution.
  • Such a system has broad applications in diverse biological processes. Given that RNA modifications are shared among all eukaryotic cells and RNA viruses that replicate inside cell nuclei, RNA modifications have been actively studied in cancer, immunology, RNA virology, etc. In addition, the spatial distribution of these modifications is likely important in fields including neuroscience, stem cell differentiation, and developmental biology.
  • the methods described herein also have the potential to provide an integrative transcriptomic and epitranscriptomic spatial cell atlas of the brain that cannot be charted by existing approaches and new scientific knowledge on how RNA modifications regulate cell functions across different cell types and how single-cell events collectively impact tissue function.
  • mRNA modifications e.g., 1 -methyladenosine, pseudouridine, etc.
  • tRNAs have a plethora of diverse modifications. 3 Increasing evidence indicates that they can also impact protein translation in response to various signals, stresses, and human diseases. 3,5
  • N 6 -methyladenosine (m 6 A) is the most prevalent internal modification present in the messenger RNA of all higher eukaryotes. This modification is non-stoichiometric and is installed by the m 6 A methyltransferase complex METTL3/14 (“writers”) whose deficiency is lethal in vertebrates; its presence is further dynamically regulated by m 6 A demethylases (ALKBH5/FTO; “erasers”) which impact development, fertility, and nutrient metabolism.
  • m 6 A-binding proteins, YTHDFs and YTHDCs specifically recognize m 6 A- modified mRNAs and regulate mRNA splicing, export, translation, and degradation. 12,13 Together, the m 6 A regulatory proteins endow gene expression with fast response and controllable protein production, essential during stem cell differentiation and animal development 3,14 .
  • m 6 A-sequencing of rodents has revealed that m 6 A is prevalent in the whole brain transcriptome, modifying thousands of coding genes and hundreds of non-coding genes (FIG. 6). 15,16 Studies of m 6 A pathway in animals have suggested that m 6 A modulates neuronal functions, including corticogenesis, dopaminergic signaling in the mouse midbrain, flight and locomotive behaviors in flies, neurogenesis in adult mice, and axon regeneration in mice. Upregulation of m 6 A has been observed to occur with brain maturation, behavioral experience, and memory formation, suggesting a link between m 6 A accumulation and brain activity.
  • ADHD attention- deficit/hyperactivity disorder
  • MDD major depression disorder
  • addiction epilepsy
  • neurodegeneration m 6 A methyltransferases and demethylases have been associated with attention- deficit/hyperactivity disorder (ADHD), major depression disorder (MDD), addiction, epilepsy, and neurodegeneration. 17
  • a method for 3D-m 6 A-seq was developed by utilizing m 6 A-specific binders, proximity amplification, and in situ RNA sequencing (FIG. 7): (1) m 6 A-binders (anti-m 6 A antibodies or biochemically purified m 6 A-specific YTH domain proteins 12 13 ) are conjugated with a DNA primer and used to stain samples of interest; (2) DNA padlock probes are then used to hybridize to the RNA sequence in proximity to m 6 A sites.
  • RNA-templated DNA ligase e.g., SplintR
  • the circularized DNA probes close to m 6 A sites are then amplified by rolling circle amplification (RCA, thousands of tandem repeats), while those near unmethylated sites cannot be amplified due to a lack of complementary primers attached to m 6 A binders needed for circularization
  • each padlock probe contains a 5-base or longer barcode to encode gene identity (>1000 genes) and another 1-base barcode to encode m 6 A-site identity within the transcript (5 rounds of sequencing allows for up to 20 sites per transcript; >99% m 6 A modified genes have ⁇ 20 sites per transcript); and (6) the gene identity and m 6 A-site identity are read out by 6 rounds of combinatorial SEDAL sequencing and 5 rounds sequential SED
  • 3D-m 6 A-seq is a collection of single RNA molecules resolved with gene identity, 3D coordinates, and the number/location of m 6 A peaks.
  • Such data can be directly visualized and analyzed to uncover the spatial distribution of gene expression with subcellular resolution (approximately 150-400 nm spatial resolution, depending on both the size of DNA amplicons and optical limits 11 ).
  • cell-body staining e.g., Nissl Staining
  • cell segmentation the RNAs can be attributed to each cell.
  • the data can then be analyzed for simultaneous cell-type classification, evaluation of singlecell variation of gene expression, and m 6 A methylation status.
  • 3D-m 6 A-seq can be applied to study the two most established biological systems for molecular-level investigation of neural activity: potassium chloride (KC1) depolarization of primary neuronal cell cultures and the dark/light conditioning of mice (FIG. 8) 11 .
  • Samples can be collected at a series of time points pre- and post-stimulation to trace the spatiotemporal dynamics and single-cell diversity of the m 6 A epitranscriptome in response to global neuron stimulation, evaluating the distribution of m 6 A RNAs in immediate-early genes (IEG) versus late-response genes (LRG).
  • IEG immediate-early genes
  • LRG late-response genes
  • Example 3 Three-dimensional in situ profiling of single-cell epitranscriptomics
  • the transcriptome serves as the pivotal mechanism that transmits information from the upstream genome to the downstream proteome, regulating numerous cellular processes.
  • single-cell RNA sequencing and spatial transcriptomic techniques have successfully mapped the transcriptomes of all kinds of biological samples, revealing intricate cell-to-cell heterogeneity in various systems.
  • additional information beyond the RNA sequence is embedded in the transcriptome.
  • This additional information termed the epitranscriptome, namely includes the chemical modifications on mRNA, such as N 6 - methyladenosine (m 6 A), and the RNA-binding proteins (RBPs) associated with them, which are crucial in regulating almost every stage of the mRNA life cycle, ranging from mRNA synthesis, translocation, translation, and degradation.
  • m 6 A-map in situ m 6 A mapping
  • 3D three-dimensional
  • m 6 A is the most abundant and arguably the most important modification on mRNA, which has been reported to regulate mRNA translation efficiency, stability, translocation, and splicing (FIG. 9A). Moreover, m 6 A is also implicated in many biological processes at tissue level, including learning and memory, cancer progression, and neurodegeneration. Thus, it is likely that different cells exhibit heterogeneous m 6 A methylomes and utilize m 6 A in different ways to regulate their transcriptome.
  • m 6 A methylomes For instance, what exactly the cell-type/state-specific m 6 A methylomes are, whether the same cell types can be divided into granular subtypes based on m 6 A methylome, whether m 6 A- mRNA and non-m 6 A-mRNAs exhibit different localizations at subcellular level and tissue level, how heterogeneous m 6 A affects different kinetics of RNA life cycle in different cell types, and many other such questions remain unanswered (FIG. 9C).
  • the circularized padlock probes were amplified using in situ rolling circle amplification (RCA).
  • RCA in situ rolling circle amplification
  • amino-allyl-dUTP was spiked in the RCA reaction so that the amplification products (amplicons) would be functionalized with primary amine groups, which were further acryloylated by methacrylic acid N- hydroxysuccinimide ester (MA-NHS) and embedded in a polyacrylamide hydrogel network.
  • MA-NHS methacrylic acid N- hydroxysuccinimide ester
  • the barcodes on padlock probes were in situ sequenced, achieving multiplexity up to thousands of genes (FIG. 10A).
  • PAPG may be replaced with a secondary antibody.
  • the secondary antibody can be conjugated to the oligo using, for example, the SiteClick antibody labeling kit (FIG. 11A).
  • m 6 A-map vl was tested with rabbit secondary antibody (FIG. 1 IB).
  • An antibody-independent biotinylated- YTH-streptavidin-oligo detection scheme was tested as well (FIG. 10D).
  • the method can also be optimized in multiple ways. For example: (1) the optimal position of the probes was found to be 10 nt from m 6 A sites of interest; (2) rRNA blocking probes, which can hybridize to rRNA sequences containing m 6 A sites, can be added to the hybridization mixture to reduce m 6 A signals from rRNA m 6 A sites that are false positives; and (3) anchoring mRNAs to the hydrogel before hybridization followed by proteinase treatment can allow for better antibody diffusion into the sample (e.g., EDC that preferentially reacts with RNAs can be used to attach a polymerizable handle to the RNA).
  • EDC that preferentially reacts with RNAs can be used to attach a polymerizable handle to the RNA
  • m 6 A-map vl can only detect m 6 A-modified RNA but cannot detect unmethylated RNA, it is very valuable to simultaneously detect both methylated and unmethylated RNA given the following considerations: (1) classification of cell states or cell types using marker gene expression; (2) estimation of m 6 A stoichiometry to distinguish whether a higher m 6 A signal from a certain gene is due to a higher expression level or a higher m 6 A fraction; (3) comparison between the differential localization of methylated RNA and unmethylated RNA; and (4) integration with other in situ sequencing data. An updated method, m 6 A-map v2, was devised to address this issue.
  • the steps before the second hybridization are the same as m 6 A-map vl.
  • a second hybridization using STARmap probes is performed, which allows for the detection of non-m 6 A RNAs, followed by ligation and the second RCA (FIG. 12A). If there is already a DNA amplicon attached to the RNA, the second RCA will not be able to occur because the first DNA amplicon has taken up most of the available space (FIG. 12B). Both m 6 A and non-m 6 A RNAs can thereby be quantified separately, and the fraction of m 6 A RNAs can be calculated. Quantification of the m 6 A fraction at single m 6 A sites (ACTB and MAEAT1) in cell culture was demonstrated (FIGs. 12C and 12D). The m 6 A fractions at different cell cycle phases were indeed different, and m 6 A RNAs and non-m 6 A RNAs showed distinct subcellular localization.
  • RBP-RNA interaction is another critical aspect of the epitranscriptome since most mRNA modifications achieve their functions via differential binding of various RBPs.
  • RBP-RNA interactome mapping methods have been developed, these methods are either tailored for detecting ribosome- mRNA interaction specifically or require transfection of exogenous gene constructs.
  • m 6 A sometimes has contradictory functions depending on context, and the main reason is that it may be bound by different reader RBPs that trigger completely different downstream pathways.
  • a multiplexed detection method would be ideal for interpretation. Therefore, an in situ multiplexed RBP-RNA mapping technology was developed, which includes transferring the information of oligo-conjugated antibodies to the padlock probe followed by in situ sequencing of transferred barcode information (FIG. 13A). The feasibility of single RBP-RNA mapping has already been demonstrated (FIG. 13B), and the next step is to multiplex this method (FIG. 13C). Such a method is useful for determining how epitranscriptome and RBP-RNA interaction contributes to cell- state/type- specific transcriptome regulation and tissue functions.
  • m 6 A-map can detect multiple m 6 A sites simultaneously in a high-throughput manner
  • FUCCI Fluorescent Ubiquitination-based Cell Cycle Indicator
  • Spatial transcriptomic profiling (STARmap) and m 6 A-map vl were performed simultaneously.
  • Antibodies from different vendors were tested in order to select the one with the highest detection efficiency.
  • the nuclei were stained with DAPI, while the cell bodies were stained with Flamingo and the endoplasmic reticulum (ER) were stained with concanavalin A, which enabled visualization of probes for three moderately-expressed housekeeping genes (HMBS, MRPL19, and PGK1).
  • HMBS, MRPL19, and PGK1 moderately-expressed housekeeping genes
  • the in situ sequencing data and FUCCI imaging data of m 6 A-map vl was preprocessed by deconvolution, spot finding, read assignment, cellular and subcellular segmentation, alignment, filtering, and normalization.
  • the number of reads of m 6 A-map is lower than that in STARmap (FIGs. 15A and 15C).
  • the Invitrogen RM362 anti-m 6 A antibody achieved the best signal-to-noise ratio (SNR), reaching an SNR of 25 for most of the genes (FIG. 15B).
  • m 6 A-map a novel strategy, m 6 A-map, was developed that can be used to measure hundreds or thousands of m 6 A loci in single cells with subcellular resolution and estimate the relative stoichiometry for each locus in a semi-quantitative manner.
  • m 6 A-map can be used to discover brand new biological insights related to the epitranscriptome, such as whether different epitranscriptomic status affects the subcellular localization of RNA, how the epitranscriptome exhibits cell-to-cell heterogeneity, and whether m 6 A levels fluctuate in different cell cycle phases.
  • the methods described herein are versatile tools for exploring m 6 A biology in situ for cell culture and tissue samples.
  • the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features.

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